Engineered polypeptides and uses thereof

ABSTRACT

Polypeptides, such as antibody molecules and fusion proteins, comprising an Fc region, are disclosed. The polypeptides can be used to treat, prevent, and/or diagnose disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/667,309, filed Aug. 2, 2017, which claims the benefit of U.S.Provisional Application No. 62/370,201, filed Aug. 2, 2016, and U.S.Provisional Application No. 62/485,671, filed Apr. 14, 2017. Thecontents of the aforesaid applications are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 1, 2017, isnamed P2029-701410_SL.txt and is 3,446 bytes in size.

BACKGROUND

Monoclonal antibody therapies are a class of immunotherapies thatinvolve monoclonal antibodies (mAbs) that are capable of specificallyinteracting with disease-relevant biological molecules. In recent years,the disease areas that therapeutic antibodies can target havesignificantly expanded, and a number of monoclonal antibodies andantibody-derivative products have been approved for therapeutic use inthe United States and many other countries. Monoclonal antibodytherapies are currently used or investigated for treating variousdiseases or conditions, including, for example, infectious diseases,cancer, immune diseases, organ transplantation, cardiovascular diseases,and metabolic diseases.

The efficacy of monoclonal antibodies can be achieved by differentmechanisms of action (Suzuki et al. J Toxicol Pathol. 2015; 28(3):133-139). Many therapeutic antibodies neutralize the pathophysiologicalfunction of their target molecules or cells. In recent years, monoclonalantibodies that block immune checkpoints have been used to enhanceantitumor immunity in cancer patients with the potential to producedurable clinical responses. Certain monoclonal antibodies can triggerantibody-dependent cell-mediated cytotoxic (ADCC) activity orcomplement-dependent cytotoxic (CDC) activity. Monoclonal antibodies canalso be used as drug delivery carriers, for example, when conjugated toradioisotopes, toxins, or other therapeutic or diagnostic agents.

Given the ability of monoclonal antibodies and antibody-derivativeproducts in modulating various biological functions, the need exists fordeveloping new approaches for generation of polypeptides (e.g., antibodymolecules or fusion proteins) suitable for treating, preventing, anddiagnosing disorders.

SUMMARY

This disclosure provides, at least in part, polypeptides (e.g., antibodymolecules or fusion proteins) that comprise an Fc region of animmunoglobulin, and that comprise one or more of the structural orfunctional properties disclosed herein. In an embodiment, nucleic acidmolecules encoding the polypeptides, expression vectors, host cells,compositions (e.g., pharmaceutical compositions), kits, containers, andmethods for making the polypeptides (e.g., antibody molecules or fusionproteins), are also provided. The polypeptides (e.g., antibody moleculesor fusion proteins) disclosed herein can be used (alone or incombination with other agents or therapeutic modalities) to treat,prevent, and/or diagnose disorders, such as disorders and conditionsdisclosed herein.

In an aspect, the disclosure features a polypeptide, e.g., an antibodymolecule or fusion protein, comprising an Fc region, wherein the Fcregion comprises a mutation, and wherein the polypeptide has one or more(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all) of the followingproperties:

a) has an increased binding affinity (e.g., a decreased dissociationconstant (K_(d))) for a neonatal Fc receptor (FcRn), e.g., at a pHbetween 6.0 and 6.5 (e.g., at pH 6.0), e.g., at least 1.5, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, or 50-fold increase, compared to areference polypeptide, e.g., as determined by an octet-based assay or acell-based assay;

b) has a higher binding affinity (e.g., a lower dissociation constant(K_(d))) for an FcRn at a pH between 6.0 and 6.5 (e.g., at pH 6.0),e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 50-foldhigher, than the binding affinity at a pH between 7.0 and 7.4 (e.g., atpH 7.4), e.g., as determined by an octet-based assay or a cell-basedassay;

c) binds to an FcRn with high affinity, e.g., at a pH between 6.0 and6.5 (e.g., at pH 6.0), e.g., with a dissociation constant (K_(d)) of 300nM or less, e.g., 250 nM or less, 200 nM or less, 150 nM or less, 100 nMor less, 50 nM or less, e.g., 25 nM or less, 10 nM or less, 5 nM orless, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.2 nM or less, 0.1 nMor less, 0.05 nM or less, 0.02 nM or less, or 0.01 nM or less, e.g.,between 25 nM and 0.1 nM, between 20 nM and 0.5 nM, between 15 nM and 1nM, between 10 nM and 5 nM, or between 20 nM and 10 nM, e.g., asdetermined by an octet-based assay or a cell-based assay;

d) binds to an FcRn with low affinity e.g., at a pH between 7.0 and 7.4(e.g., at pH 7.4), e.g., with a K_(d) of 50 nM or more, e.g., 60 nM ormore, 80 nM or more, 100 nM or more, 150 nM or more, 200 nM or more, 500nM or more, e.g., between 50 nM and 500 nM or between 100 nM and 250 nM,e.g., as determined by an octet-based assay or a cell-based assay;

e) has the same binding affinity, does not substantially alter (e.g.,decreases or increases by no more than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%) the binding affinity, or increases the bindingaffinity (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or50-fold), for an Fcγ receptor (e.g., one, two, or all of FcγRI,FcγRIIa/b, or FcγRIII), compared to a reference polypeptide, e.g., asdetermined by an octet-based assay or a cell-based assay;

f) has the same thermal stability, or does not substantially alter(e.g., increases or decreases the melting temperature by no more than 1°C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C.),the thermal stability, compared to a reference polypeptide, e.g., asdetermined by a sypro orange assay;

g) has the same binding affinity, does not substantially alter (e.g.,decreases or increases by no more than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%) the binding affinity, or increases the bindingaffinity (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or50-fold), for C1q, compared to a reference polypeptide, e.g., asdetermined by ELISA;

h) has the same binding affinity, does not substantially alter (e.g.,decreases or increases by no more than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%) the binding affinity, or increases the bindingaffinity (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or50-fold), for TRIM21, compared to a reference polypeptide, e.g., asdetermined by ELISA;

i) has the same effector function, or does not substantially alter(e.g., decreases or increases by more than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%) an effector function, or increases an effectorfunction (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or50-fold), e.g., one or more (e.g., two, three, or all) of a complementdependent cytotoxicity (CDC), an antibody dependent cell mediatedcytotoxicity (ADCC), an antibody dependent cell mediated phagocytosis(ADCP), or an antibody dependent intracellular neutralization (ADIN),compared to a reference polypeptide;

j) has an increased half-life in vivo, e.g., at least 1.5, 2, 3, 4, 5,6, 7, 8, 9, or 10-fold increase, compared to a reference polypeptide,e.g., as determined in an animal model;

k) has the same biological function, does not substantially alter (e.g.,decreases or increases by no more than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%) a biological function, or increases a biologicalfunction (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or50-fold), in vitro, ex vivo, or in vivo, compared to a referencepolypeptide;

l) has the same developability characteristic, does not substantiallyalter (e.g., decreases or increases by no more than 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90%) a developability characteristic, orincreases a developability characteristic (e.g., by at least 1.5, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 50-fold), e.g., one or more (e.g., two,three, or all) of stability, solubility, aggregation, or expressionlevel, compared to a reference polypeptide;

m) has the same binding affinity, specificity, or both, or does notsubstantially alter (e.g., decreases or increases by no more than 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) the binding affinity,specificity, or both, or increases the binding affinity, specificity, orboth (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or50-fold), for an epitope, compared to a reference polypeptide; or

n) increases mucosal uptake, e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, or 50-fold, compared to a reference polypeptide,e.g., as determined by a transcytosis assay.

In an embodiment, the polypeptide has an increased binding affinity(e.g., a decreased dissociation constant (K_(d))) for a neonatal Fcreceptor (FcRn), e.g., at a pH between 6.0 and 6.5 (e.g., at pH 6.0),e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or50-fold increase, compared to a reference polypeptide, e.g., asdetermined by an octet-based assay or a cell-based assay.

In an embodiment, the polypeptide has a higher binding affinity (e.g., alower dissociation constant (K_(d))) for an FcRn at a pH between 6.0 and6.5 (e.g., at pH 6.0), e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, or 50-fold higher, than the binding affinity at a pH between 7.0and 7.4 (e.g., at pH 7.4), e.g., as determined by an octet-based assayor a cell-based assay.

In an embodiment, the polypeptide binds to an FcRn with high affinitye.g., at a pH between 6.0 and 6.5 (e.g., at pH 6.0), e.g., with adissociation constant (K_(d)) of 50 nM or less, e.g., 25 nM or less, 10nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less,0.2 nM or less, 0.1 nM or less, 0.05 nM or less, 0.02 nM or less, or0.01 nM or less, e.g., between 25 nM and 0.1 nM, between 20 nM and 0.5nM, between 15 nM and 1 nM, between 10 nM and 5 nM, or between 20 nM and10 nM, e.g., as determined by an octet-based assay or a cell-basedassay.

In an embodiment, the polypeptide binds to an FcRn with low affinitye.g., at a pH between 7.0 and 7.4 (e.g., at pH 7.4), e.g., with a K_(d)of 50 nM or more, e.g., 60 nM or more, 80 nM or more, 100 nM or more,150 nM or more, 200 nM or more, 500 nM or more, e.g., between 50 nM and500 nM or between 100 nM and 250 nM, e.g., as determined by anoctet-based assay or a cell-based assay.

In an embodiment, the polypeptide has the same binding affinity, doesnot substantially alter (e.g., decreases or increases by no more than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) the binding affinity, orincreases the binding affinity (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 50-fold), for an Fcγ receptor (e.g., one, two, orall of FcγRI, FcγRIIa/b, or FcγRIII), compared to a referencepolypeptide, e.g., as determined by an octet-based assay or a cell-basedassay.

In an embodiment, the polypeptide has the same thermal stability, ordoes not substantially alter (e.g., increases or decreases the meltingtemperature by no more than 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7°C., 8° C., 9° C., or 10° C.), the thermal stability, compared to areference polypeptide, e.g., as determined by a sypro orange assay.

In an embodiment, the polypeptide has the same binding affinity, doesnot substantially alter (e.g., decreases or increases by no more than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) the binding affinity, orincreases the binding affinity (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 50-fold), for C1q, compared to a referencepolypeptide, e.g., as determined by ELISA.

In an embodiment, the polypeptide has the same binding affinity, doesnot substantially alter (e.g., decreases or increases by no more than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) the binding affinity, orincreases the binding affinity (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 50-fold), for TRIM21, compared to a referencepolypeptide, e.g., as determined by ELISA.

In an embodiment, the polypeptide has the same effector function, ordoes not substantially alter (e.g., decreases or increases by more than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) an effector function, orincreases an effector function (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 50-fold), e.g., one or more (e.g., two, three, orall) of a complement dependent cytotoxicity (CDC), an antibody dependentcell mediated cytotoxicity (ADCC), an antibody dependent cell mediatedphagocytosis (ADCP), or an antibody dependent intracellularneutralization (ADIN), compared to a reference polypeptide.

In an embodiment, the polypeptide has an increased half-life in vivo,e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold increase,compared to a reference polypeptide, e.g., as determined in an animalmodel.

In an embodiment, the polypeptide has the same biological function, doesnot substantially alter (e.g., decreases or increases by no more than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) a biological function,or increases a biological function (e.g., by at least 1.5, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, or 50-fold), in vitro, ex vivo, or in vivo,compared to a reference polypeptide. In an embodiment, the biologicalfunction comprises an inhibitory (e.g., neutralizing) activity. In anembodiment, the biological function comprises inhibiting (e.g.,neutralizing) a pathogen, e.g., a virus, a bacterium, or a fungus. In anembodiment, the biological function comprising an anti-tumor activity.In an embodiment, the biological function comprises inhibiting an immuneresponse. In an embodiment, the biological function comprises anagonistic activity. In an embodiment, the biological function comprisesactivating or restoring an immune response.

In an embodiment, the polypeptide has the same developabilitycharacteristic, does not substantially alter (e.g., decreases orincreases by no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90%) a developability characteristic, or increases a developabilitycharacteristic (e.g., by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, or 50-fold), e.g., one or more (e.g., two, three, or all) ofstability, solubility, aggregation, or expression level, compared to areference polypeptide;

In an embodiment, the polypeptide has the same binding affinity,specificity, or both, or does not substantially alter (e.g., decreasesor increases by no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90%) the binding affinity, specificity, or both, or increases thebinding affinity, specificity, or both (e.g., by at least 1.5, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, or 50-fold), for an epitope, compared to areference polypeptide.

In an embodiment, the polypeptide increases mucosal uptake, e.g., by atleast 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50-fold,compared to a reference polypeptide, e.g., as determined by atranscytosis assay.

In an embodiment, the polypeptide has properties a) or b) above. In anembodiment, the polypeptide has properties a) and b) above. In anembodiment, the polypeptide has one or both of properties a) or b)above, and one or both of properties c) or d) above. In an embodiment,the polypeptide has one or both of properties a) or b) above, and one,two, three, four, or all of properties e), f), g), h), or i) above. Inan embodiment, the polypeptide has one or both of properties a) or b)above, and one or both of properties c) or d) above. In an embodiment,the polypeptide has one or both of properties a) or b) above, and one,two, three, four, five, six, or all of properties c), d), j), k), l),m), or n) above. In an embodiment, the polypeptide has one or both ofproperties a) or b) above, one, two, three, four, or all of propertiese), f), g), h), or i) above, and one, two, three, four, five, six, orall of properties c), d), j), k), l), m), or n) above. In an embodiment,the polypeptide has one, two, three, or all of properties a), b), c), ord) above, one, two, three, four, or all of properties e), f), g), h), ori) above, and one, two, three, four, or all of properties j), k), l),m), or n) above. In an embodiment, the polypeptide has one or both ofproperties a) or c) above, one or both of properties b) or d) above,one, two, three, four, or all of properties e), f), g), h), or i) above,and one, two, three, four, or all of properties j), k), l), m), or n)above.

In an embodiment, the reference polypeptide is an otherwise identicalpolypeptide without the mutation, e.g., comprising a wild-type Fcregion, e.g., having the amino acid sequence of SEQ ID NO: 1, or anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, or which differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 15 amino acid residues.

In an embodiment, the mutation is in a residue in a CH2 domain Inanother embodiment, the mutation is in a residue in a CH3 domain In anembodiment, the polypeptide comprises at least one mutation in a residuein a CH2 domain and at least one mutation in a residue in a CH3 domainIn an embodiment, the polypeptide further comprises a mutation in aresidue in a region other than a CH2 domain and/or a CH3 domain.

In an embodiment, the mutation does not alter, or does not substantiallyalter, the conformation of the linker region between the CH2 and CH3domains. In an embodiment, the mutation does not introduce a cluster(e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more consecutive) of hydrophobic oraromatic residues, e.g., on a surface region (e.g., a region defined asthe area covered by amino acid residues that have more than 20% solventaccessible area).

In an embodiment, the polypeptide is an antibody molecule, e.g., anantibody molecule described herein.

In an embodiment, the polypeptide is an IgG, e.g., IgG1, IgG2, IgG3, orIgG4. In an embodiment, the polypeptide is an IgG1. In an embodiment,the polypeptide is an IgG4.

In an embodiment, the polypeptide comprises a heavy chain immunoglobulinvariable region, a light chain immunoglobulin variable region, or both.In an embodiment, the polypeptide comprises a tetramer of two heavychain immunoglobulin variable regions and two light chain immunoglobulinvariable regions. In an embodiment, the polypeptide comprises a fulllength antibody molecule. In an embodiment, the polypeptide comprises afragment (e.g., an antigen-binding fragment) of an antibody molecule.

In an embodiment, the polypeptide comprises a chimeric antibodymolecule. In an embodiment, the polypeptide comprises a humanizedantibody molecule. In an embodiment, the polypeptide comprises a humanantibody molecule. In an embodiment, the polypeptide comprises a murineantibody molecule. In an embodiment, the polypeptide comprises abispecific or multispecific antibody molecule.

In another embodiment, the polypeptide is a fusion protein, e.g., afusion protein described herein. In an embodiment, the polypeptidecomprises a fragment (e.g., functional fragment) of the fusionpolypeptide.

In an embodiment, the polypeptide comprises one or more (e.g., 2, 3, 4,or all) of the following:

(i) a mutation in a residue in a surface region (e.g., a region definedas the area covered by amino acid residues that have more than 20%solvent accessible area) that interacts with the FcRn, e.g., an FcRncontact residue;

(ii) a mutation in a residue that is a peripheral residue along theFc-FcRn interface (e.g., any amino acid residues on the surface of theFc region that is less than 7 Angstroms from the FcRn in the Fc-FcRncomplex);

(iii) a mutation is in a residue that is non-contact residue in Fc-FcRnbinding;

(iv) a mutation in a residue which is a helix contact reside thatenhances the conformational dynamics of 250-helix, e.g., a helixcomprising one or more (e.g., 2, 3, 4, 5, or all) of P247, K248, D249,T250, L251, or M252; or

(v) a mutation, which modulates pK of a histidine and/or is anintroduction of a histidine along the Fc-FcRn interface (e.g., any aminoacid residues on the surface of the Fc region that is less than 7Angstroms from the FcRn in the Fc-FcRn complex).

In an embodiment, the polypeptide comprises (i) and (ii) above. In anembodiment, the polypeptide comprises (i) and (iii) above. In anembodiment, the polypeptide comprises (i) and (iv) above. In anembodiment, the polypeptide comprises (i) and (v) above. In anembodiment, the polypeptide comprises (ii) and (iii) above. In anembodiment, the polypeptide comprises (ii) and (iv) above. In anembodiment, the polypeptide comprises (ii) and (v) above. In anembodiment, the polypeptide comprises (iii) and (iv) above. In anembodiment, the polypeptide comprises (iii) and (v) above. In anembodiment, the polypeptide comprises (iv) and (v) above.

In an embodiment, the polypeptide comprises (i), (ii) and (iii) above.In an embodiment, the polypeptide comprises (i), (ii) and (iv) above. Inan embodiment, the polypeptide comprises (i), (ii) and (v) above. In anembodiment, the polypeptide comprises (i), (iii) and (iv) above. In anembodiment, the polypeptide comprises (i), (iii) and (iv) above. In anembodiment, the polypeptide comprises (i), (iv) and (v) above. In anembodiment, the polypeptide comprises (ii), (iii) and (iv) above. In anembodiment, the polypeptide comprises (ii), (iii) and (v) above. In anembodiment, the polypeptide comprises (ii), (iv) and (v) above. In anembodiment, the polypeptide comprises (iii), (iv) and (v) above.

In an embodiment, the polypeptide comprises (i), (ii), (iii) and (iv)above. In an embodiment, the polypeptide comprises (i), (ii), (iii) and(v) above. In an embodiment, the polypeptide comprises (i), (ii), (iv)and (v) above. In an embodiment, the polypeptide comprises (i), (iii),(iv) and (v) above. In an embodiment, the polypeptide comprises (ii),(iii), (iv) and (v) above.

In an embodiment, the polypeptide comprises (i), (ii), (iii), (iv), and(v) above.

In an embodiment, the polypeptide comprises a mutation in a residue in asurface region (e.g., a region defined as the area covered by amino acidresidues that have more than 20% solvent accessible area) that interactswith the FcRn, e.g., an FcRn contact residue.

In an embodiment, the mutation is in a residue chosen from: L251, 1253,R255, P257, H285, N286, K288, T307, V308, L309, Q311, L314, H310, H433,N434, H435, or Y436. In an embodiment, the polypeptide comprises aplurality of mutations in two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, or all) of the residues chosen from L251, 1253,R255, P257, H285, N286, K288, T307, V308, L309, Q311, L314, H310, H433,N434, H435, or Y436.

In an embodiment, the polypeptide comprises a mutation in a residue thatis a peripheral residue along the Fc-FcRn interface (e.g., any aminoacid residues on the surface of the Fc region that is less than 7Angstroms from the FcRn in the Fc-FcRn complex).

In an embodiment, the mutation is in a residue chosen from one or more(e.g., 2, 3, 4, 5, 6, or all) of T256, H285, N286, T307, Q311, N315, orA378. In an embodiment, the polypeptide comprises a plurality ofmutations in two or more (e.g., 2, 3, 4, 5, 6, or all) of the residueschosen from T256, H285, N286, T307, Q311, N315, or A378. In anembodiment, the polypeptide comprises one or more (e.g., 2, 3, 4, 5, 6,or all) of the mutations chosen from T256D, H285N, N286D, T307Q, Q311V,N315D, or A378V.

In an embodiment, the mutation is in a residue chosen from T256, Q311,or A378. In an embodiment, the polypeptide comprises a plurality ofmutations in two or all of the residues chosen from T256, Q311, or A378.In an embodiment, the polypeptide comprises one, two, or all of themutations chosen from T256D, Q311V, or A378V.

In an embodiment, the mutation is in a residue chosen from H285, T307,or N315. In an embodiment, the polypeptide comprises a plurality ofmutations in two or all of the residues chosen from H285, T307, or N315.In an embodiment, the polypeptide comprises one, two, or all of themutations chosen from H285N, T307Q, or N315D.

In an embodiment, the mutation is in a residue chosen from H285, T307,or A378. In an embodiment, the polypeptide comprises a plurality ofmutations in two or all of the residues chosen from H285, T307, or A378.In an embodiment, the polypeptide comprises one, two, or all or themutations chosen from H285D, T307Q, or A378V.

In an embodiment, the mutation is in a residue chosen from T307, Q311,or A378. In an embodiment, the polypeptide comprises a plurality ofmutations in two or all of the residues chosen from T307, Q311, or A378.In an embodiment, the polypeptide comprises one, two, or all of themutations chosen from T307Q, Q311V, or A378V.

In an embodiment, the mutation is in a residue chosen from T256, N286,T307, Q311, or A378. In an embodiment, the polypeptide comprises aplurality of mutations in two, three, four, or all of the residueschosen T256, N286, T307, Q311, or A378. In an embodiment, thepolypeptide comprises one, two, three, four, or all of the mutationschosen from T256D, N286D, T307R, Q311V, or A378V.

In an embodiment, the mutation is in a residue chosen from T256, H285,T307, Q311, or A378. In an embodiment, the polypeptide comprises aplurality of mutations in two, three, four, or all of the residueschosen T256, H285, T307, Q311, or A378. In an embodiment, thepolypeptide comprises one, two, three, four, or all of the mutationschosen from T256D, H285D, T307R, Q311V, or A378V.

In an embodiment, the mutation is in a residue chosen from M252, T256,T307, L309, Q311, H433, N434, Y436, N286, or K288. In an embodiment, thepolypeptide comprises a plurality of mutations in two or more (e.g., 3,4, 5, 6, 7, 8, 9, or all) of the residues chosen from M252, T256, T307,L309, Q311, H433, N434, Y436, N286, or K288.

In an embodiment, the polypeptide comprises a mutation is in a residuethat is non-contact residue in Fc-FcRn binding.

In an embodiment, the mutation is in a residue chosen from A287, V308,N315, L314, L432, H429, E430, or A431. In an embodiment, the polypeptidecomprises a plurality of mutations in two or more (e.g., 3, 4, 5, 6, 7,or all) of the residues chosen from A287, V308, N315, L314, L432, H429,E430, or A431.

In an embodiment, the polypeptide comprises a mutation in a residuewhich is a helix contact reside that enhances the conformationaldynamics of 250-helix (e.g., a helix comprising one or more (e.g., 2, 3,4, 5, or all) of P247, K248, D249, T250, L251, or M252), e.g., a lateraldisplacement or conformational flexibility exhibited by the 250-helix(e.g., as shown by a comparison of the crystal structures of the Fcdomain crystallized at pH5.0 (PDB ID: 4J12) and at pH 6.5 (PDB ID:4Q7D).

In an embodiment, the mutation is in a residue chosen from P244, P245,T250, L251, P247, E380, M428, A378, D376, P257, V308, A287, L306, orH427. In an embodiment, the polypeptide comprises a plurality ofmutations in two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, orall) of the residues chosen from P244, P245, T250, L251, P247, E380,M428, A378, D376, P257, V308, A287, L306, or H427.

In an embodiment, the polypeptide comprises a mutation which is theintroduction of a histidine along the Fc-FcRn interface (e.g., any aminoacid residues on the surface of the Fc region that is less than 7Angstroms from the FcRn in the Fc-FcRn complex).

In an embodiment, the mutation introduces a polar amino acid residue. Inanother embodiment, the mutation introduces a non-polar amino acidresidue. In an embodiment, the mutation introduces a charged amino acidresidue. In another embodiment, the mutation introduces a non-chargeamino acid residue. In an embodiment, the mutation introduces apositively charged (or basic) amino acid residue. In another embodiment,the mutation introduces a negatively charged (or acidic) amino acidresidue. In an embodiment, the mutation introduces a hydrophobic aminoacid residue. In another embodiment, the mutation introduces ahydrophilic amino acid residue.

In an embodiment, the polypeptide comprises a mutation in at least one(e.g., 2, 3, 4, 5, or more) FcRn contact residue. In an embodiment, thepolypeptide comprises a mutation in one or more (e.g., 2 or all) ofresidues T256, T307, or N286. In an embodiment, the polypeptidecomprises mutations in residues T256 and T307, optionally, furthercomprises a mutation in residue N286. In an embodiment, the mutation inresidue T256 is a polar residue, e.g., chosen from T256D, T256E, orT256R. In an embodiment, the mutation in residue T256 is T256D. In anembodiment, the mutation in residue T307 is a polar residue, e.g.,chosen from T307D, T307E, or T307R. In an embodiment, the mutation inresidue T307 is T307R. In an embodiment, the mutation in residue N286 isN286I. In an embodiment, the polypeptide comprises mutations T256D andT307R. In an embodiment, the polypeptide comprises mutations T256D,T307R, and N286I.

In an embodiment, the polypeptide comprises a mutation in at least one(e.g., 2, 3, 4, 5, or more) FcRn contact residue. In an embodiment, thepolypeptide comprises a mutation in one or more (e.g., 2 or all) ofresidues L309, D312, or N315. In an embodiment, the polypeptidecomprises a mutation in residue L309, optionally, further comprisesmutations in residues D312 and N315. In an embodiment, the mutation inresidue L309 is L309N. In an embodiment, the mutation in residue D312 isD312A. In an embodiment, the mutation in residue N315 is N315D. In anembodiment, the polypeptide comprises mutation L309N. In an embodiment,the polypeptide comprises mutations L309N, D312A and N315D.

In an embodiment, the polypeptide comprises a mutation in at least one(e.g., 1, 2, 3, 4, 5, or more) FcRn non-contact residue. In anembodiment, the polypeptide comprises a mutation in one or more (e.g., 2or all) of residues L209R, D312, or Q311. In an embodiment, thepolypeptide comprises mutations in residues L309 and D312, optionally,further comprising a mutation in residue Q311. In an embodiment, thepolypeptide comprises a mutation in residue Q311. In an embodiment, themutation in residue L309 is L309R. In an embodiment, the mutation inresidue D312 is D312E. In an embodiment, the mutation in residue Q311 isQ311P. In an embodiment, the polypeptide comprises mutations L309R andD312E. In an embodiment, the polypeptide comprises mutations L309R,D312E, and Q311P. In an embodiment, the polypeptide comprises mutationQ311P.

In an embodiment, the polypeptide comprises a mutation in at least one(e.g., 2, 3, 4, 5, or more) FcRn contact residue. In an embodiment, thepolypeptide comprises a mutation in one or more (e.g., 2, 3, or 4) ofresidues chosen from 1253, 5254, M252, or R255. In an embodiment, thepolypeptide comprises a mutation in residue 1253. In an embodiment, themutation in residue 1253 is I253M. In an embodiment, the polypeptidecomprises a mutation in residue 5254. In an embodiment, the mutation inresidue 5254 is S254H or S254M. In an embodiment, the polypeptidecomprises mutations in residues M252 and 5254. In an embodiment, themutation in residue M252 is M252E. In an embodiment, the mutation inresidue 5254 is S254R. In an embodiment, the polypeptide comprisesmutations in residues M252, 5254, and R255. In an embodiment, themutation in residue M252 is M252E. In an embodiment, the mutation inresidue 5254 is S254R. In an embodiment, the mutation in residue R255 isR255Y.

In an embodiment, the polypeptide comprises a mutation in at least one(e.g., 2, 3, 4, 5, or more) FcRn non-contact residue. In an embodiment,the polypeptide comprises mutations that are equivalent of T250Q andM34L. In an embodiment, the polypeptide comprises a mutation in one ormore (e.g., 2, 3, 4, or all) of residues chosen from D376, K248, E380,M428, or A328. In an embodiment, the polypeptide comprises a mutation inresidue D376 and a mutation in a residue chosen from K248, E380, M428,or A328. In an embodiment, the polypeptide comprises a mutation inresidue D376 and a mutation in residue K248. In an embodiment, thepolypeptide comprises a mutation in residue D376 and a mutation inresidue E380. In an embodiment, the polypeptide comprises a mutation inresidue D376 and a mutation in residue M428. In an embodiment, thepolypeptide comprises a mutation in residue D376 and a mutation inresidue A328. In an embodiment, the mutation in residue D376 is D376Q orD376N. In an embodiment, the mutation in residue K248 is K248S. In anembodiment, the mutation in residue E380 is E380A. In an embodiment, themutation in residue D376 is D376Q. In an embodiment, the mutation inresidue M428 is M428L. In an embodiment, the mutation in residue A328 isA328I. In an embodiment, the polypeptide comprises mutation D376Q orD376N, and mutation K248S. In an embodiment, the polypeptide comprisesmutation D376Q or D376N, and mutation E380A. In an embodiment, thepolypeptide comprises mutations D376Q and M428L. In an embodiment, thepolypeptide comprises mutations D376Q and A328I.

In an embodiment, the polypeptide comprises a mutation in at least one(e.g., 2, 3, 4, 5, or more) FcRn non-contact residue. In an embodiment,the polypeptide comprises a mutation in one or more (e.g., 2 or all) ofresidues chosen from K246, P247, or D376. In an embodiment, thepolypeptide comprises a mutation in residue K246 and a mutation inresidue P247, optionally, further comprising a mutation in residue D376.In an embodiment, the mutation in residue K246 is K246N. In anembodiment, the mutation in residue P247 is P247A. In an embodiment, themutation in residue D376 is D376N. In an embodiment, the polypeptidecomprises mutations K246N and P247A, optionally, further comprisingmutation D376N.

In an embodiment, the polypeptide comprises a mutation in one or more(e.g., 2, 3, 4, 5, 6, or all) of residues chosen from T256, T307, N286,A287, P257, Q311, or P247. In an embodiment, the polypeptide comprisesmutations in residues T256, T307, N286, and A287. In an embodiment, thepolypeptide comprises mutations in residues T256, T307, and P257. In anembodiment, the polypeptide comprises mutations in residues T256, T307,and Q311. In an embodiment, the polypeptide comprises mutations inresidues T256, T307, and P247. In an embodiment, mutation in residueT256 is T256D. In an embodiment, the mutation in residue T307 is T307R.In an embodiment, the mutation in residue N286 is N286I. In anembodiment, the mutation in residue A287 is A287S. In an embodiment, themutation in residue P257 is P257L. In an embodiment, the mutation inresidue Q311 is Q311V or Q311L. In an embodiment, the mutation inresidue P247 is P247D. In an embodiment, the polypeptide comprisesmutations T256D, T307R, N286D, and A287S. In an embodiment, thepolypeptide comprises mutations T256D, T307R, and P257L. In anembodiment, the polypeptide comprises mutations T256D, T307R, and Q311Vor Q311L. In an embodiment, the polypeptide comprises mutations T256D,T307R, and P247D.

In an embodiment, the polypeptide comprises a mutation in one or more(e.g., 2, 3, 4, 5, 6, 7, or all) of residues chosen from N286, A287,P247, Q311, V308, P257, N315, or V279. In an embodiment, the polypeptidecomprises mutations in residues N286, A287, P247, and Q311. In anembodiment, the mutation in residue N286 is N286D. In an embodiment, themutation in residue A287 is A287S. In an embodiment, the mutation inresidue P247 is P247D. In an embodiment, the mutation in residue Q311 isQ311V. In an embodiment, the polypeptide comprises mutations in residuesV308 and P257. In an embodiment, the mutation in residue V308 is V308N.In an embodiment, the mutation in residue P257 is P257M. In anembodiment, the polypeptide comprises mutations in residues Q311, N315,and V279. In an embodiment, the mutation in residue Q311 is Q311L. In anembodiment, the mutation in residue N315 is N315T. In an embodiment, themutation in residue V279 is V279I.

In an embodiment, the polypeptide comprises a mutation in one or more(e.g., 2, 3, or all) of residues chosen from G433 or H433, P434 or G434,G434a, or H435. In an embodiment, the polypeptide comprises mutations inresidues G433, P434, and H435. In an embodiment, the polypeptidecomprises mutations in residues G433, P434, G434a, and H435. In anembodiment, the polypeptide comprises mutations in residues H433, G434,P434a, and H435.

In an embodiment, the polypeptide comprises one or more (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, or more) mutations, or one or more combination ofmutations, as described in Table 1. For example, a combination ofmutations can include one or more mutations listed under the name FcMutXand one or more mutations listed under the name FcMutY, where X and Yare three-digit numbers shown in Table 1, and X is not equal to Y.

In an embodiment, the polypeptide comprises mutation I253M. In anembodiment, the polypeptide comprises mutations L309H, D312A and N315D.In an embodiment, the polypeptide comprises mutation L309N. In anembodiment, the polypeptide comprises mutations M252E and S254R. In anembodiment, the polypeptide comprises mutations M252E, S254R and R255Y.In an embodiment, the polypeptide comprises mutation S254H. In anembodiment, the polypeptide comprises mutation S254M. In an embodiment,the polypeptide comprises mutations T256D and T307R. In an embodiment,the polypeptide comprises mutations T256L, N286I and T3071. In anembodiment, the polypeptide comprises mutations T256I, N286I and T3071.In an embodiment, the polypeptide comprises mutations K248S and D376Q.In an embodiment, the polypeptide comprises mutations K248S and D376N.In an embodiment, the polypeptide comprises mutations D376Q and E380A.In an embodiment, the polypeptide comprises mutations D376N and E380A.In an embodiment, the polypeptide comprises mutations D376Q and M428L.In an embodiment, the polypeptide comprises mutations K248S and A378I.In an embodiment, the polypeptide comprises mutation L314K. In anembodiment, the polypeptide comprises mutation M252W. In an embodiment,the polypeptide comprises mutation V308F. In an embodiment, thepolypeptide comprises mutations V308F and N434Y. In an embodiment, thepolypeptide comprises mutations T256D, T307R and D376N. In anembodiment, the polypeptide comprises mutations L309R and D312E. In anembodiment, the polypeptide comprises mutations L309R, Q311P and D312E.In an embodiment, the polypeptide comprises mutations K246N and P247A.In an embodiment, the polypeptide comprises mutations K246N, P247A andD376N. In an embodiment, the polypeptide comprises mutations T256E andT307R. In an embodiment, the polypeptide comprises mutations T256R andT307D. In an embodiment, the polypeptide comprises mutations T256R andT307E. In an embodiment, the polypeptide comprises mutation Q311P. In anembodiment, the polypeptide comprises mutation D376Q. In an embodiment,the polypeptide comprises mutations T256D, N286D, A287S and T307R. In anembodiment, the polypeptide comprises mutations T256D, P257L and T307R.In an embodiment, the polypeptide comprises mutations T256D, T307R andQ311V. In an embodiment, the polypeptide comprises mutations P247D,T256D and T307R. In an embodiment, the polypeptide comprises mutationsP247D, N286D, A287S and Q311V. In an embodiment, the polypeptidecomprises mutations P257M and V308N. In an embodiment, the polypeptidecomprises mutations V279I, Q311L and N315T. In an embodiment, thepolypeptide comprises mutations H433G and N434P. In an embodiment, thepolypeptide comprises mutations T256D, N286D and T307R. In anembodiment, the polypeptide comprises mutations T256D, N286E and T307R.In an embodiment, the polypeptide comprises mutations T256D, N286Q andT307R. In an embodiment, the polypeptide comprises mutations T256D,P257T and T307R. In an embodiment, the polypeptide comprises mutationsT256D, P257V and T307R. In an embodiment, the polypeptide comprisesmutations T256D, T307R and Q311I. In an embodiment, the polypeptidecomprises mutations T256D, T307R and Q311L. In an embodiment, thepolypeptide comprises mutations T256D, T307R and Q311M. In anembodiment, the polypeptide comprises mutations T256D, P257L, N286D,T307R and Q311V. In an embodiment, the polypeptide comprises mutationsT256D, T307R and M428L

In an embodiment, the mutation is other than M252Y, S254T, T256E, L309N,T250Q, M428L, N434S, N434A, T307A, E380A, N434A, M252Y, S254T, T256E, ora combination thereof. In an embodiment, the mutation is in a residueother than residue M252, 5254, T256, L309, T250, M428, N434, N434, T307,E380, N434, M252, 5254, T256, or a combination thereof. In anembodiment, the polypeptide does not have one or more (e.g., 2, 3, 4, 5,6, 7, 8, 9, or all) of the following mutation or mutations: (i) M252Y,S254T, and T256E; (ii) L309N; (iii) T250Q and M428L; (iv) M428L andN434A; (v) N434A; (vi) T307A, E380A, and N434A; (vii) M252W; (viii)V308F; (ix) V308F and N434Y; or (x) H435A.

In an embodiment, the polypeptide comprises a first mutation chosen fromM252Y, S254T, T256E, L309N, T250Q, M428L, N434S, N434A, T307A, E380A,N434A, M252Y, S254T, or T256E, and a second mutation chosen from amutation in Table 1 other than M252Y, S254T, T256E, L309N, T250Q, M428L,N434S, N434A, T307A, E380A, N434A, M252Y, S254T, and T256E.

In an embodiment, the polypeptide comprises a combination of mutationschosen from M252Y, S254T, T256E, L309N, T250Q, M428L, N434S, N434A,T307A, E380A, N434A, M252Y, S254T, or T256E, wherein the combination isother than (i) M252Y, S254T, and T256E; (ii) L309N; (iii) T250Q andM428L; (iv) M428L and N434A; (v) N434A; (vi) T307A, E380A, and N434A;(vii) M252W; (viii) V308F; (ix) V308F and N434Y; or (x) H435A.

In an embodiment, the polypeptide further comprises a mutation in the Fcregion that increases an effector function. In an embodiment, themutation is in a residue chosen from 5239 (e.g., S239D), A330 (e.g.,A330L), 1332 (e.g., 1332E), F243 (e.g., F243L), G236 (e.g., G236A), or acombination thereof, e.g., to increase an effector function.

In an embodiment, the polypeptide further comprises a mutation in the Fcregion that decreases an effector function. In an embodiment, themutation is in a residue chosen from K322 (e.g., K322A), L234 (e.g.,L234A or L234F), L235 (e.g., L235A or L235E), P331 (e.g., P331S), N297,or a combination thereof, e.g., to decrease an effector function.

In an embodiment, the polypeptide further comprises a mutation in aregion other than the Fc region, e.g., in a Fab region.

In an embodiment, the polypeptide further comprises a plurality ofmutations, wherein at least one mutation is a compensating mutation,e.g., a compensating mutation described herein.

In an embodiment, the polypeptide is an isolated polypeptide. In anembodiment, the polypeptide is a synthetic polypeptide.

In an aspect, the disclosure features a composition, e.g.,pharmaceutical composition, comprising a polypeptide described herein.In an embodiment, the composition further comprises a pharmaceuticalacceptable carrier.

In an aspect, the disclosure features a nucleic acid molecule encoding apolypeptide described herein. In an aspect, the disclosure features avector comprising a nucleic acid molecule described herein. In anaspect, the disclosure features a cell, e.g., an isolated cell,comprising a nucleic acid molecule described herein or a vectordescribed herein. In an aspect, the disclosure features a kit comprisinga polypeptide described herein and instructions to use of thepolypeptide. In an aspect, the disclosure features a containercomprising a polypeptide described herein.

In an aspect, the disclosure features a method of producing apolypeptide described herein, the method comprising culturing a celldescribed herein under conditions that allow production of an antibodymolecule, thereby producing the polypeptide. In an embodiment, themethod further comprises isolating or purifying the polypeptide.

In an aspect, the disclosure features a method of treating a disorder(e.g., a disorder described herein), the method comprising administeringto a subject in need thereof an effective amount of a polypeptidedescribed herein or a composition described herein, thereby treating thedisorder.

In an aspect, the disclosure features a polypeptide described herein foruse in a method of treating a disorder (e.g., a disorder describedherein). In another aspect, the disclosure features use of a polypeptidedescribed herein in the manufacture of a medicament in the treatment ofa disorder (e.g., a disorder described herein).

In an aspect, the disclosure features a method of detecting a molecule,the method comprising contacting a cell or a sample from a subject withpolypeptide described herein, thereby detecting the molecule.

The disclosure contemplates all combinations of any one or more of theforegoing aspects and/or embodiments, as well as combinations with anyone or more of the embodiments set forth in the detailed description andexamples.

Other features, objects, and advantages of the compositions and methodsherein will be apparent from the description and drawings, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the binding between FcRn and IgG1.

FIG. 2 depicts various binding sites in the Fc region.

FIG. 3 depicts the structural basis for pH specific engagement of FcRn.

FIG. 4 depicts mapping of Fc-FcRn interaction interface. CH2 and CH3domains are indicated and shown by different gray shades.

FIG. 5 depicts a network view of Fc-FcRn engagement.

FIG. 6 depicts FcRn binding to anti-CH1 immobilized antibody and pHspecific enhancement.

FIG. 7 depicts the results of a cell-based FcRn binding competitionassay.

FIG. 8 depicts the binding of exemplary antibody molecules to FcγRI andFcγRIIIa.

FIG. 9 depicts the thermal stability of exemplary antibody molecules.

FIG. 10 depicts the half-lives of exemplary antibody molecules in Tg32mice.

FIG. 11A depicts K_(d), k_(on) and k_(off) of exemplary Fc engineeredantibodies. Values presented as fold change as compared to IgGcontaining WT Fc.

FIG. 11B depicts improved binding to FcRn at pH 6.0 and poor binding toFcRn at pH 7.4 of a representative Fc engineered antibody.

FIG. 11C depicts the interaction of the CH2 domain of the antibody Fcwith the FcRn molecule.

FIG. 12 depicts biophysical properties of Fc engineered variants.

FIG. 13A depicts improved in vivo half-life of 3 Motavizumab Fc variantsin comparison to WT Motavizumab (Mota-WT).

FIG. 13B depicts pharmacokinetic properties of 2 Motavizumab Fc variantsand WT Motavizumab.

FIG. 14A depicts improved in vivo half-life of later stage MotavizumabFc variants in comparison to WT Motavizumab (Mota-WT) administered at adose of 5 mg/kg.

FIG. 14B depicts pharmacokinetic properties of later stage MotavizumabFc variants and WT Motavizumab administered at a dose of 5 mg/kg.

FIG. 14C depicts improved in vivo half-life of later stage MotavizumabFc variants in comparison to WT Motavizumab (Mota-WT) administered at adose of 2 mg/kg.

FIG. 14D depicts pharmacokinetic properties of later stage MotavizumabFc variants and WT Motavizumab administered at a dose of 2 mg/kg.

FIG. 15 depicts longer in vivo half-life of the ZVB Zika antibody.

FIG. 16 depicts a similar half-life of ZKB-LS (ZVB antibody with LS Fcmutation) and ZKB-156 (ZVB antibody with Visterra 156 Fc mutation). Bothof these antibodies had a longer half-life than the parental ZVBantibody (ZKB) and WT Motavizumab.

FIG. 17A depicts the binding of exemplary Fc variants to FcγRI, FcγRIIA,FcγRIIB, FcγRIIIA, and C1q.

FIG. 17B depicts the binding of FcMut213 to FcγRIIIA compared towild-type.

FIG. 17C depicts the binding of FcMut213 to FcγRIIA compared towild-type.

FIG. 17D depicts the binding of FcMut213 to C1q compared to wild-type.

FIG. 18 depicts the CDC activity of exemplary Fc variants (RituximabFab). Calculation is based on concentration that achieves 50% lysis, notthe mid-point of the four-parameter fit.

FIGS. 19A-19B depict the ADCC activity of exemplary Fc variants(Rituximab Fab).

FIG. 20A depicts the IgG Fc structure and Fc-FcRn interaction.

FIG. 20B depicts the structures of Fc at two different pHs.

FIG. 21A depicts the types of residues with potential to mediate Fc-FcRninteraction.

FIG. 21B depicts the molecular interaction of key residues of exemplaryengineered Fc variants.

FIG. 22 depicts an exemplary Octet sensogram.

FIG. 23 depicts the SEC profile and Tm of exemplary Fc variants.

FIG. 24 depicts the binding of Fc variants to FcγRIIIa (ADCC activity)and to C1q (CDC activity).

FIG. 25A depicts the pharmacokinetic characterization of IgGs with Fcvariants. Plot shows change in concentration of IgG over time.

FIG. 25B depicts the PK characterization of exemplary Fc variants.

FIG. 26 depicts binding of motavizumab Fab with Fc variants to TRIM21.

FIG. 27A depicts the structures of the FcRn-Fc complex, Protein A-Fccomplex, Trim21-Fc complex, and Fc-Fc hexamer complex.

FIG. 27B depicts the superposition of crystal structures of Fc domainhighlighting Fc domain dynamics.

DETAILED DESCRIPTION

Disclosed herein are polypeptides (e.g., antibody molecules or fusionproteins) that bind to a target molecule or cell, e.g., a human proteinor cell, with high affinity and specificity. Advantageously, several ofthe polypeptides (e.g., antibody molecules or fusion proteins) describeherein have one or more improved or desired pharmacokinetic properties,such as circulating half-life. Without wishing to be bound theory, it isbelieved that polypeptides can have a range of circulating half-lives inhumans, and circulating half-life can affect, e.g., interaction withserum and cell components, rate of fluid phase pinocytosis, interactionwith FcRn, receptor mediated endocytosis, drug doses, and generation ofanti-drug antibodies. Nucleic acid molecules encoding the polypeptides(e.g., antibody molecules or fusion proteins), expression vectors, hostcells, compositions (e.g., pharmaceutical compositions), kits,containers, and methods for making the polypeptides (e.g., antibodymolecules or fusion proteins), are also provided. The polypeptides(e.g., antibody molecules or fusion proteins) and pharmaceuticalcompositions disclosed herein can be used (alone or in combination withother agents or therapeutic modalities) to treat, prevent, and/ordiagnose disorders and conditions, e.g., disorders and conditionsassociated with a target molecule (e.g., protein) or cell, e.g., adisorder or condition described herein.

Without wishing to be bound by theory, it is believed that in someembodiments, the long circulating half-life of IgGs is attributed to itsability to minimize its endosomal degradation by associating with theneonatal Fc receptor (FcRn). FcRn plays an important role in placentaltransfer of IgG molecules from mother to fetus and in serum IgGhomeostasis (Leach et al., J Immunol, 1996. 157(8): 3317-22; Simister etal., Eur J Immunol, 1996. 26(7): 1527-31; Kristoffersen, APMIS Suppl,1996. 64: 5-36; Roopenian et al., J Immunol, 2003. 170(7): 3528-33;Junghans and Anderson, Proc Natl Acad Sci USA, 1996. 93(11): 5512-6).The acidic environment of the early endosome can allow for binding ofIgG and albumin to FcRn, which protects the IgG from undergoingdegradation and helps trafficking the IgG back to the extracellularenvironment, where, upon exposure to physiological pH, the molecules arereleased back into circulation. This pathway can be largely responsiblefor the prolonged serum half-life of both IgG and albumin (Junghans andAnderson, Proc Natl Acad Sci USA, 1996. 93(11): 5512-6; Chaudhury etal., J Exp Med, 2003. 197(3): 315-22).

The Fc domain of an antibody is primarily responsible for binding toFcRn to facilitate antibody recycling. In some embodiments, antibodieswith identical Fc regions can have different circulating half-lives, atleast in part, because a number of factors such as thermal stability,interaction with serum and cell components, presence of anti-drugantibodies, high dose, receptor mediated endocytosis, and fluid phasepinocytosis, can promote antibody degradation and negatively influenceits half-life. The interaction of IgG with FcRn can serve to protect theantibody from endosomal degradation and extend the half-life of theantibody. Modification of Fc can be used to promote FcRn interaction andtherefore extend the half-life of antibodies (Ghetie et al., NatBiotechnol, 1997. 15(7): p. 637-40; Dall'Acqua et al., J Biol Chem,2006. 281(33): 23514-24; Hinton et al., J Biol Chem, 2004. 279(8):6213-6; Vaccaro et al., Nat Biotechnol, 2005. 23(10): 1283-8; Zalevskyet al., Nat Biotechnol, 2010. 28(2): 157-9; Dall'Acqua et al., JImmunol, 2002. 169(9): 5171-80; Monnet et al., MAbs, 2014. 6(2): 422-36;Monnet et al., Front Immunol, 2015. 6: 39; Shields et al., J Biol Chem,2001. 276(9): 6591-604; Robbie et al., Antimicrob Agents Chemother,2013. 57(12): 6147-53).

The Fc of IgG can also bind to various other receptors such as FcγRI,FcγRIIa, FcγRIIb, FcγRIII, C1q, and TRIM21, and these interactionsmediate various effector functions such as antibody dependent cellularcytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibodydependent cellular phagocytosis (ADCP), and antibody dependentintracellular neutralization (ADIN). Traditional approaches used toidentify the Fc variants have largely relied on random mutagenesis anddisplay formats and often compromise certain important attributes of theantibody, be it effector functions or biophysical stability.

Without wishing to be bound by theory, it is believed that in someembodiment, the engineering of Fc for FcRn binding or half-lifeextension as disclosed herein is performed in the context of the variouseffector functions mediated by Fc. For example, a structural and networkbased framework can be used to interrogate the interaction of Fc withFcRn at neutral and acidic pH. Using this framework, different pathwaysfor improving FcRn binding, e.g., decreasing the k_(off) of interaction,can be identified. The interaction networks of mutations can be mappedand mutations can be combined and assessed for binding to FcRn and otherFc receptors. For example, Fc variants that confer enhancement inhalf-life and retain and in some cases enhance effector functions suchas ADCC and CDC can be identified. With the increasing use of antibodiesand fusion proteins as therapeutics for prevention and treatment ofdifferent diseases, there have been greater needs to develop antibodiesand fusion proteins with long half-life, e.g., to treat or preventchronic diseases.

Definitions

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

The compositions and methods disclosed herein encompass polypeptides andnucleic acids having the sequences specified, or sequences substantiallyidentical or similar thereto, e.g., sequences at least 85%, 90%, 95%identical or higher to the sequence specified.

In the context of an amino acid sequence, the term “substantiallyidentical” is used herein to refer to a first amino acid that contains asufficient or minimum number of amino acid residues that are i)identical to, or ii) conservative substitutions of aligned amino acidresidues in a second amino acid sequence such that the first and secondamino acid sequences can have a common structural domain and/or commonfunctional activity. For example, amino acid sequences that contain acommon structural domain having at least about 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., asequence provided herein.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence,e.g., a sequence provided herein.

The term “functional variant” refers polypeptides that have asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina typical embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, e.g., at least 40%, 50%, 60%, e.g.,at least 70%, 80%, 90%, 100% of the length of the reference sequence.The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In some embodiments, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm which has been incorporated into theGAP program in the GCG software package (available at gcg.com), usingeither a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available at gcg.com), using a NWSgapdna.CMP matrix and a gapweight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or6. One suitable set of parameters (and the one that should be usedunless otherwise specified) are a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid as described herein. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules described herein. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seencbi.nlm.nih.gov.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6×sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions 4) aresuitable conditions and the ones that should be used unless otherwisespecified.

It is understood that the molecules described herein may have additionalconservative or non-essential amino acid substitutions, which do nothave a substantial effect on their functions.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing. As used herein the term“amino acid” includes both the D- or L-optical isomers andpeptidomimetics.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms “polypeptide,” “peptide” and “protein” (if single chain) areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. The polypeptide can be isolatedfrom natural sources, can be a produced by recombinant techniques from aeukaryotic or prokaryotic host, or can be a product of syntheticprocedures. In an embodiment, the polypeptide is an antibody molecule.In another embodiment, the polypeptide is a fusion protein.

The terms “nucleic acid,” “nucleic acid sequence,” “nucleotidesequence,” or “polynucleotide sequence,” and “polynucleotide” are usedinterchangeably. They refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. The polynucleotide may be either single-stranded ordouble-stranded, and if single-stranded may be the coding strand ornon-coding (antisense) strand. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Thesequence of nucleotides may be interrupted by non-nucleotide components.A polynucleotide may be further modified after polymerization, such asby conjugation with a labeling component. The nucleic acid may be arecombinant polynucleotide, or a polynucleotide of genomic, cDNA,semisynthetic, or synthetic origin which either does not occur in natureor is linked to another polynucleotide in a non-natural arrangement.

The term “isolated,” as used herein, refers to material that is removedfrom its original or native environment (e.g., the natural environmentif it is naturally occurring). For example, a naturally-occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or polypeptide, separated by humanintervention from some or all of the co-existing materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of the environment in which it is found in nature.

As used herein, the term “treat,” e.g., a disorder described herein,means that a subject (e.g., a human) who has a disorder, e.g., adisorder described herein, and/or experiences a symptom of a disorder,e.g., a disorder described herein, will, in an embodiment, suffer less asevere symptom and/or recover faster when an antibody molecule isadministered than if the antibody molecule were never administered.Treatment can, e.g., partially or completely, alleviate, ameliorate,relieve, inhibit, or reduce the severity of, and/or reduce incidence,and optionally, delay onset of, one or more manifestations of theeffects or symptoms, features, and/or causes of the disorder. In anembodiment, treatment is of a subject who does not exhibit certain signsof the disorder, and/or of a subject who exhibits only early signs ofthe disorder. In an embodiment, treatment is of a subject who exhibitsone or more established signs of a disorder. In an embodiment, treatmentis of a subject diagnosed as suffering from a disorder.

As used herein, the term “prevent,” a disorder, means that a subject(e.g., a human) is less likely to have the disorder, if the subjectreceives a polypeptide (e.g., antibody molecule).

Various aspects of the compositions and methods herein are described infurther detail below. Additional definitions are set out throughout thespecification.

Antibody Molecules

Disclosed herein are antibody molecules, e.g., antibody moleculescomprising an Fc region, e.g. an Fc region having one or more mutationsdescribed herein, and/or having one or more structural or functionalproperties described herein.

In an embodiment, the antibody molecule is engineered or derived from anantibody molecule (e.g., a parental antibody molecule) that contains anFc region. For example, the engineered antibody molecule, or antibodymolecule derivative, can have a different Fc region than the parentalantibody molecule. In another embodiment, the antibody molecule isengineered or derived from an antibody molecule (e.g., a parentalantibody molecule) that does not contain an Fc region. For example, theengineered antibody molecule, or antibody molecule derivative, can havean Fc region, directly or indirectly, fused to the parental antibodymolecule or a functional fragment thereof.

As used herein, the term “antibody molecule” refers to a protein, e.g.,an immunoglobulin chain or a fragment thereof, comprising at least oneimmunoglobulin variable domain sequence. The term “antibody molecule”includes, for example, full-length, mature antibodies andantigen-binding fragments of an antibody. For example, an antibodymolecule can include a heavy (H) chain variable domain sequence(abbreviated herein as VH), and a light (L) chain variable domainsequence (abbreviated herein as VL). In another example, an antibodymolecule includes two heavy (H) chain variable domain sequences and twolight (L) chain variable domain sequence, thereby forming two antigenbinding sites, such as Fab, Fab′, F(ab′)₂, Fc, Fd, Fd′, Fv, single chainantibodies (scFv for example), single variable domain antibodies,diabodies (Dab) (bivalent and bispecific), and chimeric (e.g.,humanized) antibodies, which may be produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAtechnologies. These functional antibody fragments retain the ability toselectively bind with their respective antigen or receptor. Antibodiesand antibody fragments can be from any class of antibodies including,but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass(e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The antibody moleculescan be monoclonal or polyclonal. The antibody molecule can also be ahuman, humanized, CDR-grafted, or in vitro generated antibody. Theantibody molecule can have a heavy chain constant region chosen from,e.g., IgG1, IgG2, IgG3, or IgG4. The antibody molecule can also have alight chain chosen from, e.g., kappa or lambda. The term“immunoglobulin” (Ig) is used interchangeably with the term “antibody”herein.

Examples of antigen-binding fragments include: (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a diabody(dAb) fragment, which consists of a VH domain; (vi) a camelid orcamelized variable domain; (vii) a single chain Fv (scFv), see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody.These antibody fragments may be obtained using any suitable method,including several conventional techniques known to those with skill inthe art, and the fragments can be screened for utility in the samemanner as are intact antibodies.

The term “antibody” includes intact molecules as well as functionalfragments thereof. Constant regions of the antibodies can be altered,e.g., mutated, to modify the properties of the antibody (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,or complement function).

The antibody molecule can be a single chain antibody. A single-chainantibody (scFV) may be engineered (see, for example, Colcher, D. et al.(1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin CancerRes 2:245-52). The single chain antibody can be dimerized ormultimerized to generate multivalent antibodies having specificities fordifferent epitopes of the same target protein.

The antibody molecules disclosed herein can also be single domainantibodies. Single domain antibodies can include antibodies whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainantibodies, antibodies naturally devoid of light chains, single domainantibodies derived from conventional 4-chain antibodies, engineeredantibodies and single domain scaffolds other than those derived fromantibodies. Single domain antibodies may be any of the art, or anyfuture single domain antibodies. Single domain antibodies may be derivedfrom any species including, but not limited to mouse, human, camel,llama, fish, shark, goat, rabbit, and bovine. According to some aspects,a single domain antibody is a naturally occurring single domain antibodyknown as heavy chain antibody devoid of light chains. Such single domainantibodies are disclosed in WO 94/04678, for example. For clarityreasons, this variable domain derived from a heavy chain antibodynaturally devoid of light chain is known herein as a VHH or nanobody todistinguish it from the conventional VH of four chain immunoglobulins.Such a VHH molecule can be derived from antibodies raised in Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain antibodiesnaturally devoid of light chain; such VHHs are also contemplated.

The VH and VL regions can be subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR),interspersed with regions that are more conserved, termed “frameworkregions” (FR or FW). The terms “complementarity determining region,” and“CDR,” as used herein refer to the sequences of amino acids withinantibody variable regions which confer antigen specificity and bindingaffinity. As used herein, the terms “framework,” “FW” and “FR” are usedinterchangeably.

The extent of the framework region and CDRs has been precisely definedby a number of methods (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Chothia, C. etal. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used byOxford Molecular's AbM antibody modeling software. See, generally, e.g.,Protein Sequence and Structure Analysis of Antibody Variable Domains.In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R.,Springer-Verlag, Heidelberg). In an embodiment, the followingdefinitions are used: AbM definition of CDR1 of the heavy chain variabledomain and Kabat definitions for the other CDRs. In an embodiment, Kabatdefinitions are used for all CDRs. In addition, embodiments describedwith respect to Kabat or AbM CDRs may also be implemented using Chothiahypervariable loops. Each VH and VL typically includes three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may or may not include one, two, or more N- orC-terminal amino acids, or may include other alterations that arecompatible with formation of the protein structure.

The term “antigen-binding region” refers to the part of an antibodymolecule that comprises determinants that form an interface that bindsto an antigen, or an epitope thereof. With respect to proteins (orprotein mimetics), the antigen-binding region typically includes one ormore loops (of at least, e.g., four amino acids or amino acid mimics)that form an interface that binds to the antigen. Typically, theantigen-binding region of an antibody molecule includes at least one ortwo CDRs and/or hypervariable loops, or more typically at least three,four, five or six CDRs and/or hypervariable loops.

The terms “compete” or “cross-compete” are used interchangeably hereinto refer to the ability of an antibody molecule to interfere withbinding of another antibody molecule, to a target. The interference withbinding can be direct or indirect (e.g., through an allostericmodulation of the antibody molecule or the target). The extent to whichan antibody molecule is able to interfere with the binding of anotherantibody molecule to the target, and therefore whether it can be said tocompete, can be determined using a competition binding assay, forexample, a FACS assay, an ELISA or BIACORE assay. In an embodiment, acompetition binding assay is a quantitative competition assay. In anembodiment, a first antibody molecule is said to compete for binding tothe target with a second antibody molecule when the binding of the firstantibody molecule to the target is reduced by 10% or more, e.g., 20% ormore, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more,65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% ormore, 95% or more, 98% or more, 99% or more in a competition bindingassay (e.g., a competition assay described herein).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Amonoclonal antibody can be made by hybridoma technology or by methodsthat do not use hybridoma technology (e.g., recombinant methods).

An “effectively human” protein is a protein that does not evoke aneutralizing antibody response, e.g., the human anti-murine antibody(HAMA) response. HAMA can be problematic in a number of circumstances,e.g., if the antibody molecule is administered repeatedly, e.g., intreatment of a chronic or recurrent disease condition. A HAMA responsecan make repeated antibody administration potentially ineffectivebecause of an increased antibody clearance from the serum (see, e.g.,Saleh et al., Cancer Immunol. Immunother. 32:180-190 (1990)) and alsobecause of potential allergic reactions (see, e.g., LoBuglio et al.,Hybridoma, 5:5117-5123 (1986)).

The antibody molecule can be a polyclonal or a monoclonal antibody. Insome embodiments, the antibody can be recombinantly produced, e.g.,produced by any suitable phage display or combinatorial methods.

Various phage display and combinatorial methods for generatingantibodies are known in the art (as described in, e.g., Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO92/18619; Dower et al. International Publication No. WO 91/17271; Winteret al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Ladner et al. International Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contentsof all of which are incorporated by reference herein).

In an embodiment, the antibody molecule is a fully human antibody (e.g.,an antibody made in a mouse which has been genetically engineered toproduce an antibody from a human immunoglobulin sequence), or anon-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g.,monkey), camel antibody. In an embodiment, the non-human antibody is arodent (mouse or rat antibody). Methods of producing rodent antibodiesare known in the art.

Human monoclonal antibodies can be generated using transgenic micecarrying the human immunoglobulin genes rather than the mouse system.Splenocytes from these transgenic mice immunized with the antigen ofinterest are used to produce hybridomas that secrete human mAbs withspecific affinities for epitopes from a human protein (see e.g., Wood etal. International Application WO 91/00906, Kucherlapati et al. PCTpublication WO 91/10741; Lonberg et al. International Application WO92/03918; Kay et al. International Application 92/03917; Lonberg et al.1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21;Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855;Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

An antibody can be one in which the variable region, or a portionthereof, e.g., the CDRs, are generated in a non-human organism, e.g., arat or mouse. Chimeric, CDR-grafted, and humanized antibodies are withinthe invention. Antibodies generated in a non-human organism, e.g., a rator mouse, and then modified, e.g., in the variable framework or constantregion, to decrease antigenicity in a human are within the invention.

Chimeric antibodies can be produced by any suitable recombinant DNAtechnique. Several are known in the art (see Robinson et al.,International Patent Publication PCT/US86/02269; Akira, et al., EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., International Application WO 86/01533; Cabilly et al. U.S. Pat.No. 4,816,567; Cabilly et al., European Patent Application 125,023;Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.(1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005;Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. NatlCancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two butgenerally all three recipient CDRs (of heavy and or light immunoglobulinchains) replaced with a donor CDR. The antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody tolipopolysaccharide. In an embodiment, the donor will be a rodentantibody, e.g., a rat or mouse antibody, and the recipient will be ahuman framework or a human consensus framework. Typically, theimmunoglobulin providing the CDRs is called the “donor” and theimmunoglobulin providing the framework is called the “acceptor.” In someembodiments, the donor immunoglobulin is a non-human (e.g., rodent). Theacceptor framework is typically a naturally-occurring (e.g., a human)framework or a consensus framework, or a sequence about 85% or higher,e.g., 90%, 95%, 99% or higher identical thereto.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody can be humanized by any suitable method, and several suchmethods known in the art (see e.g., Morrison, S. L., 1985, Science229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen etal. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents ofall of which are hereby incorporated by reference).

Humanized or CDR-grafted antibodies can be produced by CDR-grafting orCDR substitution, wherein one, two, or all CDRs of an immunoglobulinchain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al.1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidleret al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539,the contents of all of which are hereby expressly incorporated byreference. Winter describes a CDR-grafting method which may be used toprepare humanized antibodies (UK Patent Application GB 2188638A, filedon Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of whichis expressly incorporated by reference.

Also provided are humanized antibodies in which specific amino acidshave been substituted, deleted or added. Criteria for selecting aminoacids from the donor are described in, e.g., U.S. Pat. No. 5,585,089,e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of whichare hereby incorporated by reference. Other techniques for humanizingantibodies are described in Padlan et al. EP 519596 A1, published onDec. 23, 1992.

In an embodiment, the antibody molecule has a heavy chain constantregion chosen from, e.g., the heavy chain constant regions of IgG1, IgG2(e.g., IgG2a), IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly,chosen from, e.g., the (e.g., human) heavy chain constant regions ofIgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody moleculehas a light chain constant region chosen from, e.g., the (e.g., human)light chain constant regions of kappa or lambda. The constant region canbe altered, e.g., mutated, to modify the properties of the antibodymolecule (e.g., to increase or decrease one or more of: Fc receptorbinding, antibody glycosylation, the number of cysteine residues,effector cell function, and/or complement function). In an embodiment,the antibody molecule has effector function and can fix complement. Inanother embodiment, the antibody molecule does not recruit effectorcells or fix complement. In certain embodiments, the antibody moleculehas reduced or no ability to bind an Fc receptor. For example, it may bean isotype or subtype, fragment or other mutant, which does not supportbinding to an Fc receptor, e.g., it has a mutagenized or deleted Fcreceptor binding region.

In an embodiment, a constant region of the antibody molecule is altered.Methods for altering an antibody constant region are known in the art.Antibody molecules s with altered function, e.g. altered affinity for aneffector ligand, such as FcR on a cell, or the C1 component ofcomplement can be produced by replacing at least one amino acid residuein the constant portion of the antibody with a different residue (seee.g., EP 388,151 A1, U.S. Pat. Nos. 5,624,821 and 5,648,260, thecontents of all of which are hereby incorporated by reference) Aminoacid mutations which stabilize antibody structure, such as S228P (EUnomenclature, S241P in Kabat nomenclature) in human IgG4 are alsocontemplated. Similar type of alterations could be described which ifapplied to the murine, or other species immunoglobulin would reduce oreliminate these functions.

In an embodiment, the only amino acids in the antibody molecule arecanonical amino acids. In an embodiment, the antibody molecule comprisesnaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and/or allstereoisomers of any of any of the foregoing. The antibody molecule maycomprise the D- or L-optical isomers of amino acids and peptidomimetics.

A polypeptide of an antibody molecule described herein may be linear orbranched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The antibody molecule may also bemodified; for example, by disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation,such as conjugation with a labeling component. The polypeptide can beisolated from natural sources, can be a produced by recombinanttechniques from a eukaryotic or prokaryotic host, or can be a product ofsynthetic procedures.

The antibody molecule described herein can be used alone in unconjugatedform, or can be bound to a substance, e.g., a toxin or moiety (e.g., atherapeutic drug; a compound emitting radiation; molecules of plant,fungal, or bacterial origin; or a biological protein (e.g., a proteintoxin) or particle (e.g., a recombinant viral particle, e.g., via aviral coat protein). For example, the antibody molecule can be coupledto a radioactive isotope such as an α-, β-, or γ-emitter, or a β- andγ-emitter.

An antibody molecule can be derivatized or linked to another functionalmolecule (e.g., another peptide or protein). As used herein, a“derivatized” antibody molecule is one that has been modified. Methodsof derivatization include but are not limited to the addition of afluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinityligand such as biotin. Accordingly, the antibody molecules are intendedto include derivatized and otherwise modified forms of the antibodiesdescribed herein, including immunoadhesion molecules. For example, anantibody molecule can be functionally linked (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a toxin, a pharmaceuticalagent, and/or a protein or peptide that can mediate association of theantibody or antibody portion with another molecule (such as astreptavidin core region or a polyhistidine tag).

Some types of derivatized antibody molecule are produced by crosslinkingtwo or more antibodies (of the same type or of different types, e.g., tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an anti-dengue antibody molecule maybe derivatized (or labeled) to include fluorescent compounds, variousenzymes, prosthetic groups, luminescent materials, bioluminescentmaterials, fluorescent emitting metal atoms, e.g., europium (Eu), andother anthanides, and radioactive materials (described below). Exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5dimethylamine-1-napthalenesulfonyl chloride,phycoerythrin and the like. An antibody may also be derivatized withdetectable enzymes, such as alkaline phosphatase, horseradishperoxidase, β-galactosidase, acetylcholinesterase, glucose oxidase andthe like. When an antibody is derivatized with a detectable enzyme, itis detected by adding additional reagents that the enzyme uses toproduce a detectable reaction product. For example, when the detectableagent horseradish peroxidase is present, the addition of hydrogenperoxide and diaminobenzidine leads to a colored reaction product, whichis detectable. An antibody molecule may also be derivatized with aprosthetic group (e.g., streptavidin/biotin and avidin/biotin). Forexample, an antibody may be derivatized with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Examplesof suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes luminol; and examples of bioluminescentmaterials include luciferase, luciferin, and aequorin.

Labeled antibody molecules can be used, for example, diagnosticallyand/or experimentally in a number of contexts, including (i) to isolatea predetermined antigen by standard techniques, such as affinitychromatography or immunoprecipitation; (ii) to detect a predeterminedantigen (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the protein; (iii)to monitor protein levels in tissue as part of a clinical testingprocedure, e.g., to determine the efficacy of a given treatment regimen.

An antibody molecule may be conjugated to another molecular entity,typically a label or a therapeutic (e.g., antimicrobial (e.g.,antibacterial or bactericidal), immunomodulatory, immunostimularoty,cytotoxic, or cytostatic) agent or moiety. Radioactive isotopes can beused in diagnostic or therapeutic applications. Radioactive isotopesthat can be coupled to the antibody molecules include, but are notlimited to α, β-, or γ-emitters, or β- and γ-emitters. Such radioactiveisotopes include, but are not limited to iodine (¹³¹I or ¹²⁵I), yttrium(⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium, astatine(²¹¹At), rhenium (¹⁸⁶Re), bismuth (²¹²Bi or ²¹³Bi), indium (¹¹¹In),technetium (⁹⁹mTc), phosphorus (³²P), rhodium (¹⁸⁸Rh), sulfur (³⁵S),carbon (¹⁴C), tritium (³H), chromium (⁵¹Cr), chlorine (³⁶Cl), cobalt(⁵⁷Co or ⁵⁸Co), iron (⁵⁹Fe), selenium (⁷⁵Se), or gallium (⁶⁷Ga).Radioisotopes useful as therapeutic agents include yttrium (⁹⁰Y),lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium, astatine (²¹¹At), At)rhenium (¹⁸⁶Re), bismuth (²¹²Bi or ²¹³Bi), and rhodium (¹⁸⁸Rh).Radioisotopes useful as labels, e.g., for use in diagnostics, includeiodine (¹³¹I or ¹²⁵I), indium (¹¹¹In), technetium (⁹⁹mTc), phosphorus(³²P), carbon (¹⁴C), and tritium (³H), or one or more of the therapeuticisotopes listed above.

The present disclosure provides radiolabeled antibody molecules andmethods of labeling the same. In an embodiment, a method of labeling anantibody molecule is disclosed. The method includes contacting anantibody molecule, with a chelating agent, to thereby produce aconjugated antibody. The conjugated antibody is radiolabeled with aradioisotope, e.g., ¹¹¹Indium, ⁹⁰Yttrium and ¹⁷⁷Lutetium, to therebyproduce a labeled antibody molecule.

In some aspects, this disclosure provides a method of making an antibodymolecule disclosed herein. The method includes: providing an antigen, ora fragment thereof; obtaining an antibody molecule that specificallybinds to the antigen; evaluating efficacy of the antibody molecule inmodulating activity of the antigen and/or organism expressing theantigen. The method can further include administering the antibodymolecule, including a derivative thereof (e.g., a humanized antibodymolecule) to a subject, e.g., a human.

This disclosure provides an isolated nucleic acid molecule encoding theabove antibody molecule, vectors and host cells thereof. The nucleicacid molecule includes, but is not limited to, RNA, genomic DNA andcDNA.

Fusion Proteins

Disclosed herein are fusion proteins, e.g., fusion proteins comprisingan Fc region, e.g. an Fc region having one or more mutations describedherein, and/or having one or more structural or functional propertiesdescribed herein.

In an embodiment, the fusion protein is engineered or derived from apolypeptide (e.g., a fusion protein) that contains an Fc region (e.g., aparental polypeptide). For example, the engineered polypeptide, orderivative, can have a different Fc region than the parentalpolypeptide. In another embodiment, the fusion protein is engineered orderived from a polypeptide that does not contain an Fc region (e.g., aparental polypeptide). For example, the engineered antibody molecule, orantibody molecule derivative, can have an Fc region, directly orindirectly, fused to the parental polypeptide.

As used herein, the term “fusion protein” refers to a protein,comprising two or more protein or peptide components. The two or moreprotein or peptide components can be obtained from different sources orencoded by different genes. A fusion protein is sometimes also referredto as a chimeric protein. An Fc-fusion protein (also known as Fcchimeric fusion protein, Fc-Ig, Ig-based chimeric fusion protein, orFc-tag protein) can include an Fc region of an immunoglobulin (e.g., anFc region described herein) linked (e.g., fused) to a protein orpeptide. The Fc region can be linked (e.g., fused genetically) to theprotein or peptide directly, or indirectly, e.g., through a linker. Inan embodiment, the Fc region is derived from the Fc region of IgG, e.g.,human IgG, e.g., IgG1, IgG2, IgG3, or IgG4. In an embodiment, the Fcregion is derived from the Fc region of IgG1, e.g., human IgG1.

The Fc-fused binding partner can include a variety of proteins orpeptides, or fragments thereof. For example, the Fc region can be fusedto a peptide (e.g., a therapeutic peptide), a ligand (e.g., a ligandthat activates upon binding with a cell surface receptor), a signalingmolecule, the extracellular domain of a receptor, or a bait protein(e.g., used to identify a binding partner, e.g., in a proteinmicroarray).

In an embodiment, the Fc fusion protein comprises an extracellulardomain of a receptor or a soluble receptor, or a ligand binding portionthereof. In an embodiment, the receptor is a growth factor receptor. Inan embodiment, the receptor is a cytokine receptor. In an embodiment,the receptor is an immune checkpoint molecule.

In an embodiment, the fusion protein comprises a vascular endotheliagrowth factor (VEGF)-binding portion from the extracellular domain ofhuman VEGF receptors 1 and 2, fused to the Fc region of human IgG1. Inan embodiment, the fusion protein is aflibercept. In an embodiment, thefusion protein (e.g., aflibercept) is used to treat a disorder describedherein, e.g., an eye disorder (e.g., wet macular degeneration) or acancer (e.g., a colorectal cancer).

In another embodiment, the fusion protein comprises a soluble tumornecrosis factor (TNF) receptor 2 fused to the Fc region of the humanIgG1. In an embodiment, the fusion protein is etanercept. In anembodiment, the Fc fusion protein (e.g., etanercept) is used to treat adisorder described herein, e.g., an autoimmune disorder (e.g.,rheumatoid arthritis).

In yet another embodiment, the fusion protein comprises ligand-bindingdomains of the extracellular portions of human interleukin-1 receptor 1(IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP) fused to the Fcregion of human IgG1. In an embodiment, the fusion protein isrilonacept. In an embodiment, the fusion protein (e.g., rilonacept) isused to treat a disorder described herein, e.g., a cryopyrin-associatedperiodic syndrome (CAPS), e.g., familial cold autoinflammatory syndrome,Muckle-Wells syndrome, or a neonatal onset multisystem inflammatorydisease.

In still another embodiment, the fusion protein comprises theextracellular domain of CTLA-4 fused to the Fc region of human IgG1. Inan embodiment, the fusion protein is abatacept or belatacept. In anembodiment, the fusion protein (e.g., abatacept or belatacept) is usedto treat a disorder described herein, e.g., an organ rejection, anautoimmune disorder (e.g., rheumatoid arthritis), or a cancer.

In an embodiment, the Fc fusion protein comprises a peptide, e.g., atherapeutic peptide.

In an embodiment, the fusion protein comprises a thrombopoietin-bindingpeptide fused to the Fc region of human IgG1. In an embodiment, thefusion protein is romiplostim. In an embodiment, the fusion protein(e.g., romiplostim) is used to treat a disorder described herein, e.g.,chronic idiopathic (immune) thrombocytopenic purpura (ITP).

In an embodiment, the fusion protein comprises the extracellularCD2-binding portion of the human leukocyte function antigen-3 (LFA-3)fused to the Fc region of human IgG1. In an embodiment, the fusionprotein is alefacept. In an embodiment, the fusion protein (e.g.,alefacept) is used to treat a disorder described herein, e.g., anautoimmune disorder (e.g., psoriasis) or a cancer (e.g., a cutaneousT-cell lymphoma or a T-cell non-Hodgkin lymphoma).

In an embodiment, the fusion protein comprises a coagulation factor.

In an embodiment, the fusion protein comprises Factor IX fused with theFc region of IgG1. In another embodiment, the fusion protein comprisesFactor VIII fused with the Fc region of IgG1. In an embodiment thefusion protein (e.g., the FIX-Fc fusion or FVIII-Fc fusion) is used totreat a disorder described herein, e.g., hemophilia A or hemophilia B.

In an embodiment, the fusion protein comprises one or more glycosylationsites, or is glycosylated. In another embodiment, the fusion proteindoes not have a glycosylation site, or is not glycosylated.

In an embodiment, the only amino acids in the fusion protein arecanonical amino acids. In an embodiment, the fusion protein comprisesnaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and/or allstereoisomers of any of any of the foregoing. The fusion protein maycomprise the D- or L-optical isomers of amino acids and peptidomimetics.

In an aspect, this disclosure provides a method of making a fusionprotein disclosed herein. The fusion proteins described herein can beproduced by any suitable recombinant DNA technique. In an embodiment,the method includes culturing a cell containing a nucleic acid encodingthe fusion protein under conditions that allow production of the fusionprotein. In another embodiment, the method further includes isolating orpurifying the fusion protein. In yet another embodiment, the methodfurther includes evaluating efficacy of the fusion protein in acell-based assay or in an animal model. In still another embodiment, themethod further includes administering the fusion protein to a subject,e.g., a human.

This disclosure provides an isolated nucleic acid molecule encoding theabove fusion proteins, vectors and host cells thereof. The nucleic acidmolecule includes, but is not limited to, RNA, genomic DNA and cDNA.

Fe Region

A fragment crystallizable region, or Fc region, refers to a region of animmunoglobulin that is capable of interacting with an Fc receptor. In anembodiment, the Fc region is also capable of interacting with a proteinof the complement system. While without wishing to be bound by theory,it is believed that in an embodiment, the interaction between the Fcregion with an Fc receptor, allows for activation of the immune system.

In IgG, IgA and IgD antibody isotypes, the naturally-occurring Fc regiongenerally comprises two identical protein fragments, derived from thesecond and third constant domains of the antibody's two heavy chains.Naturally-occurring IgM and IgE Fc regions generally comprise threeheavy chain constant domains (C_(H) domains 2-4) in each polypeptidechain. The Fc regions of IgGs can contain a highly conservedN-glycosylation site (Stadlmann et al. (2008). Proteomics 8 (14):2858-2871; Stadlmann (2009) Proteomics 9 (17): 4143-4153). While notwishing to be bound by theory, it is believed that in an embodiment,glycosylation of the Fc fragment contributes to Fc receptor-mediatedactivities (Peipp et al. (2008) Blood 112 (6): 2390-2399). In anembodiment, the N-glycans attached to this site are predominantlycore-fucosylated diantennary structures of the complex type. In anotherembodiment, small amounts of these N-glycans also contain bisectingGlcNAc and/or α-2,6 linked sialic acid residues.

An exemplary Fc region amino acid sequence is shown below.

(SEQ ID NO: 1) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL H N H YTQKSLSLSPGK

In SEQ ID NO: 1, the first amino acid residue in this sequence isreferred to as position 118 herein. The three Histidines in bold andunderlined are positions 310, 433 and 435, respectively.

A polypeptide (e.g., an antibody molecule or fusion protein) describedherein can have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) ofmutations or combinations of mutations described in Table 1.

TABLE 1 Exemplary Fc mutations Name Mutation FcMut001 I253M FcMut002L309H_D312A_N315D FcMut003 L309N FcMut004 M252E_S254R FcMut005M252E_S254R_R255Y FcMut006 S254H FcMut007 S254M FcMut008 T256D_T307RFcMut009 T256L_N286I_T307I FcMut010 T256I_N286I_T307I FcMut011K248S_D376Q FcMut012 K248S_D376N FcMut013 D376Q_E380A FcMut014D376N_E380A FcMut015 D376Q_M428L FcMut016 K248S_A378I FcMut017 L314KFcMut018 T250Q_M428L FcMut019 M428L_N434A FcMut020 N434A FcMut021T307A_E380A_N434A FcMut022 M252W FcMut023 V308F FcMut024 V308F_N434YFcMut026 T256D_T307R_D376N FcMut027 L309R_D312E FcMut028L309R_Q311P_D312E FcMut029 K246N_P247A FcMut030 K246N_P247A_D376NFcMut031 T256E_T307R FcMut032 T256R_T307D FcMut033 T256R_T307E FcMut034Q311P FcMut035 D376Q FcMut036 L234A_L235A FcMut037 L235V_G236A FcMut038L234P_L235P FcMut039 L235P FcMut040 P329G FcMut041 P329E FcMut042 E233KFcMut043 T256D_N286D_A287S_T307R FcMut044 T256D_P257L_T307R FcMut045T256D_T307R_Q311V FcMut046 P247D_T256D_T307R FcMut047P247D_N286D_A287S_Q311V FcMut048 P257M_V308N FcMut049 V279I_Q311L_N315TFcMut050 M428L_N434S FcMut051 N434S FcMut052 H433G_N434P FcMut053V259I_V308F_M428L FcMut067 T256D_N286D_T307R FcMut068 T256D_N286E_T307RFcMut069 T256D_N286Q_T307R FcMut070 T256D_P257T_T307R FcMut071T256D_P257V_T307R FcMut072 T256D_T307R_Q311I FcMut073 T256D_T307R_Q311LFcMut074 T256D_T307R_Q311M FcMut075 T256D_P257L_N286D_T307R_Q311VFcMut076 T256D_T307R_M428L FcMut077 M428L FcMut078 M252Y_S254T_T256QFcMut079 M252Y_S254T_T256E_K288E FcMut080 T256K_K288E FcMut081T256D_E258T FcMut082 E283Q_H285E FcMut083 R344D_D401R FcMut084K248E_E380K FcMut085 K248E_E380R FcMut086 K246H FcMut087 K248H FcMut088T250I FcMut089 T250V FcMut090 L251F FcMut091 L251M FcMut093 P257VFcMut094 N276D FcMut095 H285N FcMut096 H285D FcMut097 K288H FcMut098K288Q FcMut099 K288E FcMut100 T307E FcMut101 T307Q FcMut102 V308PFcMut103 V308I FcMut104 V308L FcMut105 L309H FcMut106 L309M FcMut107Q311H FcMut108 L314F FcMut109 Y319H FcMut110 I336T FcMut111 P343DFcMut112 P343V FcMut113 E345Q FcMut114 P346V FcMut115 P374T FcMut116D376N FcMut117 A378S FcMut118 A431T FcMut119 A431P FcMut120 A431GFcMut121 L432V FcMut122 L432I FcMut123 L432Q FcMut124 N434T FcMut125H435N FcMut126 Y436H FcMut127 K439Q FcMut128 T256D FcMut129 T307RFcMut130 A378T FcMut131 A378D FcMut132 A378H FcMut133 A378Y FcMut134A378V FcMut135 D376R FcMut136 D376F FcMut137 D376W FcMut138 L314HFcMut139 L432E_T437Q FcMut140 D376Q_A378T FcMut141 D376Q_I377M_A378TFcMut142 P244Q_D376Q FcMut143 P247T_A378T FcMut144 P247N_A378T FcMut145T256D_T307R_L309T FcMut146 A339T_S375E_F404Y FcMut147L235V_G236A_T256D_T307R FcMut148 L235V_G236A_D376Q_M428L FcMut149 L314NFcMut150 N315D FcMut151 A378T FcMut152 T437Q FcMut153 L432E FcMut154Y436R FcMut155 L314M FcMut156 L234A_L235A_T256D_T307R_Q311V FcMut157L234A_L235A_T256D_P257V_T307R FcMut158L234A_L235A_T256D_P257L_N286D_T307R_Q311V FcMut159L235V_G236A_T256D_T307R_Q311V FcMut160 L235V_G236A_T256D_P257V_T307RFcMut161 L235V_G236A_T256D_P257L_N286D_T307R_Q311V FcMut162S267T_A327N_A330M FcMut163 S267T_A327N FcMut164E235V_G236A_S267T_A327N_A330M FcMut165 F235V_G236A_S267T_A327N FcMut166M252Y_S254T FcMut167 T256E FcMut168 G236A_I332E FcMut169 S239D_I332EFcMut170 G236A_S239D_I332E FcMut171 T256D_N286D_T307R_Q311V FcMut172T256D_E258T_T307R FcMut173 T256D_E258T_T307R_Q311V FcMut174T256D_P257V_E258T_T307R FcMut175 T256D_P257L_E258T_N286D_T307R_Q311VFcMut176 T256D_E258T_N286D_T307R_Q311V FcMut177 A378V_M428L FcMut178A378V_M428I FcMut179 A378V_M428V FcMut180 T256D_N286D FcMut181T256D_A378V FcMut182 T256D_Q311V FcMut183 T256D_Q311V_A378V FcMut184T256D_T307R_A378V FcMut185 T256D_N286D_T307R_A378V FcMut186T256D_T307R_Q311V_A378V FcMut187 H285D_A378V FcMut188 H285D_Q311VFcMut189 T256D_H285D FcMut190 T256D_H285D_Q311V FcMut191T256D_H285D_T307R FcMut192 T256D_H285D_T307R_A378V FcMut193H285D_L314M_A378V FcMut194 T256D_E258T_H285D_Q311H FcMut195T256D_E258T_H285D FcMut196 H285D_N315D FcMut197 H285N_T307Q_N315DFcMut198 H285D_L432E_T437Q FcMut199 T256D_E258T_N315D FcMut200P257V_H285N FcMut201 H285N_L432F FcMut202 H285N_T437I FcMut203T256D_E258T_L314M FcMut204 T256D_E258T_T307Q FcMut205 T256D_E258T_A378VFcMut206 V308P_A378V FcMut207 P257V_A378T FcMut208 P257V_V308P_A378VFcMut209 N315D_A378T FcMut210 H285N_L314M FcMut211 L314M_L432E_T437QFcMut212 T307Q_N315D FcMut213 H285D_T307Q_A378V FcMut214 L314M_N315DFcMut215 T307Q_Q311V_A378V FcMut216 H285D_Q311V_A378V FcMut217Q311V_N315D_A378V FcMut218 T256D_E258T_Q311V FcMut219 T256D_N315D_A378VFcMut220 T256D_Q311V_N315D FcMut221 T256D_T307Q_A378V FcMut222T256D_T307Q_Q311V FcMut223 T256D_H285D_A378V FcMut224T256D_H285D_T307R_Q311V FcMut225 T256D_H285D_N286D_T307R FcMut226T256D_H285D_N286D_T307R_Q311V FcMut227 T256D_H285D_N286D_T307R_A378VFcMut228 T256D_N286D_T307R_Q311V_A378V FcMut229T256D_H285D_T307R_Q311V_A378V FcMut230 V308P_Q311V_A378V FcMut231T256D_V308P_A378V FcMut232 T256D_V308P_Q311V FcMut233 T256D_E258T_V308PFcMut234 H285D_V308P_Q311V FcMut242 E258T FcMut243 N286D FcMut244 Q311VYTE M252Y_S254T_T256E

In an embodiment, the Fc region comprises FcMut001. In an embodiment,the Fc region comprises FcMut002. In an embodiment, the Fc regioncomprises FcMut003. In an embodiment, the Fc region comprises FcMut004.In an embodiment, the Fc region comprises FcMut005. In an embodiment,the Fc region comprises FcMut006. In an embodiment, the Fc regioncomprises FcMut007. In an embodiment, the Fc region comprises FcMut008.In an embodiment, the Fc region comprises FcMut009. In an embodiment,the Fc region comprises FcMut010. In an embodiment, the Fc regioncomprises FcMut011. In an embodiment, the Fc region comprises FcMut012.In an embodiment, the Fc region comprises FcMut013. In an embodiment,the Fc region comprises FcMut014. In an embodiment, the Fc regioncomprises FcMut015. In an embodiment, the Fc region comprises FcMut016.In an embodiment, the Fc region comprises FcMut017. In an embodiment,the Fc region comprises FcMut018. In an embodiment, the Fc regioncomprises FcMut019. In an embodiment, the Fc region comprises FcMut020.In an embodiment, the Fc region comprises FcMut021. In an embodiment,the Fc region comprises FcMut022. In an embodiment, the Fc regioncomprises FcMut023. In an embodiment, the Fc region comprises FcMut024.In an embodiment, the Fc region comprises FcMut026. In an embodiment,the Fc region comprises FcMut027. In an embodiment, the Fc regioncomprises FcMut028. In an embodiment, the Fc region comprises FcMut029.In an embodiment, the Fc region comprises FcMut030. In an embodiment,the Fc region comprises FcMut031. In an embodiment, the Fc regioncomprises FcMut032. In an embodiment, the Fc region comprises FcMut033.In an embodiment, the Fc region comprises FcMut034. In an embodiment,the Fc region comprises FcMut035. In an embodiment, the Fc regioncomprises FcMut036. In an embodiment, the Fc region comprises FcMut037.In an embodiment, the Fc region comprises FcMut038. In an embodiment,the Fc region comprises FcMut039. In an embodiment, the Fc regioncomprises FcMut040. In an embodiment, the Fc region comprises FcMut041.In an embodiment, the Fc region comprises FcMut042. In an embodiment,the Fc region comprises FcMut043. In an embodiment, the Fc regioncomprises FcMut044. In an embodiment, the Fc region comprises FcMut045.In an embodiment, the Fc region comprises FcMut046. In an embodiment,the Fc region comprises FcMut047. In an embodiment, the Fc regioncomprises FcMut048. In an embodiment, the Fc region comprises FcMut049.In an embodiment, the Fc region comprises FcMut050. In an embodiment,the Fc region comprises FcMut051. In an embodiment, the Fc regioncomprises FcMut052. In an embodiment, the Fc region comprises FcMut053.In an embodiment, the Fc region comprises FcMut067. In an embodiment,the Fc region comprises FcMut068. In an embodiment, the Fc regioncomprises FcMut069. In an embodiment, the Fc region comprises FcMut070.In an embodiment, the Fc region comprises FcMut071. In an embodiment,the Fc region comprises FcMut072. In an embodiment, the Fc regioncomprises FcMut073. In an embodiment, the Fc region comprises FcMut074.In an embodiment, the Fc region comprises FcMut075. In an embodiment,the Fc region comprises FcMut076. In an embodiment, the Fc regioncomprises FcMut077. In an embodiment, the Fc region comprises FcMut078.In an embodiment, the Fc region comprises FcMut079. In an embodiment,the Fc region comprises FcMut080. In an embodiment, the Fc regioncomprises FcMut081. In an embodiment, the Fc region comprises FcMut082.In an embodiment, the Fc region comprises FcMut083. In an embodiment,the Fc region comprises FcMut084. In an embodiment, the Fc regioncomprises FcMut085. In an embodiment, the Fc region comprises FcMut086.In an embodiment, the Fc region comprises FcMut087. In an embodiment,the Fc region comprises FcMut088. In an embodiment, the Fc regioncomprises FcMut089. In an embodiment, the Fc region comprises FcMut090.In an embodiment, the Fc region comprises FcMut091. In an embodiment,the Fc region comprises FcMut093. In an embodiment, the Fc regioncomprises FcMut094. In an embodiment, the Fc region comprises FcMut095.In an embodiment, the Fc region comprises FcMut096. In an embodiment,the Fc region comprises FcMut097. In an embodiment, the Fc regioncomprises FcMut098. In an embodiment, the Fc region comprises FcMut099.In an embodiment, the Fc region comprises FcMut100. In an embodiment,the Fc region comprises FcMut101. In an embodiment, the Fc regioncomprises FcMut102. In an embodiment, the Fc region comprises FcMut103.In an embodiment, the Fc region comprises FcMut104. In an embodiment,the Fc region comprises FcMut105. In an embodiment, the Fc regioncomprises FcMut106. In an embodiment, the Fc region comprises FcMut107.In an embodiment, the Fc region comprises FcMut108. In an embodiment,the Fc region comprises FcMut109. In an embodiment, the Fc regioncomprises FcMut110. In an embodiment, the Fc region comprises FcMut111.In an embodiment, the Fc region comprises FcMut112. In an embodiment,the Fc region comprises FcMut113. In an embodiment, the Fc regioncomprises FcMut114. In an embodiment, the Fc region comprises FcMut115.In an embodiment, the Fc region comprises FcMut116. In an embodiment,the Fc region comprises FcMut117. In an embodiment, the Fc regioncomprises FcMut118. In an embodiment, the Fc region comprises FcMut119.In an embodiment, the Fc region comprises FcMut120. In an embodiment,the Fc region comprises FcMut121. In an embodiment, the Fc regioncomprises FcMut122. In an embodiment, the Fc region comprises FcMut123.In an embodiment, the Fc region comprises FcMut124. In an embodiment,the Fc region comprises FcMut125. In an embodiment, the Fc regioncomprises FcMut126. In an embodiment, the Fc region comprises FcMut127.In an embodiment, the Fc region comprises FcMut128. In an embodiment,the Fc region comprises FcMut129. In an embodiment, the Fc regioncomprises FcMut130. In an embodiment, the Fc region comprises FcMut131.In an embodiment, the Fc region comprises FcMut132. In an embodiment,the Fc region comprises FcMut133. In an embodiment, the Fc regioncomprises FcMut134. In an embodiment, the Fc region comprises FcMut135.In an embodiment, the Fc region comprises FcMut136. In an embodiment,the Fc region comprises FcMut137. In an embodiment, the Fc regioncomprises FcMut138. In an embodiment, the Fc region comprises FcMut139.In an embodiment, the Fc region comprises FcMut140. In an embodiment,the Fc region comprises FcMut141. In an embodiment, the Fc regioncomprises FcMut142. In an embodiment, the Fc region comprises FcMut143.In an embodiment, the Fc region comprises FcMut144. In an embodiment,the Fc region comprises FcMut145. In an embodiment, the Fc regioncomprises FcMut146. In an embodiment, the Fc region comprises FcMut147.In an embodiment, the Fc region comprises FcMut148. In an embodiment,the Fc region comprises FcMut149. In an embodiment, the Fc regioncomprises FcMut150. In an embodiment, the Fc region comprises FcMut151.In an embodiment, the Fc region comprises FcMut152. In an embodiment,the Fc region comprises FcMut153. In an embodiment, the Fc regioncomprises FcMut154. In an embodiment, the Fc region comprises FcMut155.In an embodiment, the Fc region comprises FcMut156. In an embodiment,the Fc region comprises FcMut157. In an embodiment, the Fc regioncomprises FcMut158. In an embodiment, the Fc region comprises FcMut159.In an embodiment, the Fc region comprises FcMut160. In an embodiment,the Fc region comprises FcMut161. In an embodiment, the Fc regioncomprises FcMut162. In an embodiment, the Fc region comprises FcMut163.In an embodiment, the Fc region comprises FcMut164. In an embodiment,the Fc region comprises FcMut165. In an embodiment, the Fc regioncomprises FcMut166. In an embodiment, the Fc region comprises FcMut167.In an embodiment, the Fc region comprises FcMut168. In an embodiment,the Fc region comprises FcMut169. In an embodiment, the Fc regioncomprises FcMut170. In an embodiment, the Fc region comprises FcMut171.In an embodiment, the Fc region comprises FcMut172. In an embodiment,the Fc region comprises FcMut173. In an embodiment, the Fc regioncomprises FcMut174. In an embodiment, the Fc region comprises FcMut175.In an embodiment, the Fc region comprises FcMut176. In an embodiment,the Fc region comprises FcMut177. In an embodiment, the Fc regioncomprises FcMut178. In an embodiment, the Fc region comprises FcMut179.In an embodiment, the Fc region comprises FcMut180. In an embodiment,the Fc region comprises FcMut181. In an embodiment, the Fc regioncomprises FcMut182. In an embodiment, the Fc region comprises FcMut183.In an embodiment, the Fc region comprises FcMut184. In an embodiment,the Fc region comprises FcMut185. In an embodiment, the Fc regioncomprises FcMut186. In an embodiment, the Fc region comprises FcMut187.In an embodiment, the Fc region comprises FcMut188. In an embodiment,the Fc region comprises FcMut189. In an embodiment, the Fc regioncomprises FcMut190. In an embodiment, the Fc region comprises FcMut191.In an embodiment, the Fc region comprises FcMut192. In an embodiment,the Fc region comprises FcMut193. In an embodiment, the Fc regioncomprises FcMut194. In an embodiment, the Fc region comprises FcMut195.In an embodiment, the Fc region comprises FcMut196. In an embodiment,the Fc region comprises FcMut197. In an embodiment, the Fc regioncomprises FcMut198. In an embodiment, the Fc region comprises FcMut199.In an embodiment, the Fc region comprises FcMut200. In an embodiment,the Fc region comprises FcMut201. In an embodiment, the Fc regioncomprises FcMut202. In an embodiment, the Fc region comprises FcMut203.In an embodiment, the Fc region comprises FcMut204. In an embodiment,the Fc region comprises FcMut205. In an embodiment, the Fc regioncomprises FcMut206. In an embodiment, the Fc region comprises FcMut207.In an embodiment, the Fc region comprises FcMut208. In an embodiment,the Fc region comprises FcMut209. In an embodiment, the Fc regioncomprises FcMut210. In an embodiment, the Fc region comprises FcMut211.In an embodiment, the Fc region comprises FcMut212. In an embodiment,the Fc region comprises FcMut213. In an embodiment, the Fc regioncomprises FcMut214. In an embodiment, the Fc region comprises FcMut215.In an embodiment, the Fc region comprises FcMut216. In an embodiment,the Fc region comprises FcMut217. In an embodiment, the Fc regioncomprises FcMut218. In an embodiment, the Fc region comprises FcMut219.In an embodiment, the Fc region comprises FcMut220. In an embodiment,the Fc region comprises FcMut221. In an embodiment, the Fc regioncomprises FcMut222. In an embodiment, the Fc region comprises FcMut223.In an embodiment, the Fc region comprises FcMut224. In an embodiment,the Fc region comprises FcMut225. In an embodiment, the Fc regioncomprises FcMut226. In an embodiment, the Fc region comprises FcMut227.In an embodiment, the Fc region comprises FcMut228. In an embodiment,the Fc region comprises FcMut229. In an embodiment, the Fc regioncomprises FcMut230. In an embodiment, the Fc region comprises FcMut231.In an embodiment, the Fc region comprises FcMut232. In an embodiment,the Fc region comprises FcMut233. In an embodiment, the Fc regioncomprises FcMut234. In an embodiment, the Fc region comprises FcMut242.In an embodiment, the Fc region comprises FcMut243. In an embodiment,the Fc region comprises FcMut244.

In an embodiment, the Fc region comprises one or more (e.g., 2, 3, 4, 5,6, 7, 8, 9, or more) of mutations or combinations of mutations chosenfrom FcMut045, FcMut171, FcMut183, FcMut186, FcMut190, FcMut197,FcMut213, FcMut215, FcMut216, FcMut219, FcMut222, FcMut223, FcMut224,FcMut226, FcMut227, FcMut228, or FcMut229. In an embodiment, the Fcregion comprises one or more (e.g., 2, 3, 4, 5, 6, or all) of mutationsor combinations of mutations chosen from FcMut045, FcMut183, FcMut197,FcMut213, FcMut215, FcMut228, or FcMut156. In another embodiment, the Fcregion comprises one or more (e.g., 2, 3, 4, 5, or all) of mutations orcombinations of mutations chosen from FcMut183, FcMut197, FcMut213,FcMut215, FcMut228, or FcMut229.

In an embodiment, the Fc region does not comprise one or more (e.g., 2,3, 4, or all) of mutations or combinations of mutations chosen fromFcMut018, FcMut021, FcMut050, FcMut102, or YTE. In an embodiment, the Fcregion comprises one or more (e.g., 2, 3, 4, or all) of mutations orcombinations of mutations chosen from FcMut018, FcMut021, FcMut050,FcMut102, or YTE, and one or more other mutations or combinations ofmutations described in Table 1.

In an embodiment, the Fc region comprises one or more (e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) ofmutations or combinations of mutations described in Table 1 that resultin a synergistic effect (e.g., binding affinity or circulatinghalf-life) as described herein.

In an embodiment, the Fc region comprises one or more (e.g., 2, 3, 4, 5,6, or 7) mutations in residues chosen from T256, H285, N286, T307, Q311,N315, or A378. In an embodiment, the Fc region comprises one or more(e.g., 2, 3, 4, 5, 6, or 7) mutations chosen from T256D, H285N, N286D,T307Q, Q311V, N315D, or A378V.

In an embodiment, the Fc region comprises a half-life enhancingmutation, a mutation that is capable of disrupting an Fc effectorfunction, or both. In an embodiment, the Fc region comprises one or moremutations or combinations of mutations described herein, e.g., chosenfrom M252W, V308F/N434Y, R255Y, P257L/N434Y, V308F, P257N/M252Y, G385N,P257N/V308Y, N434Y, M252Y/S254T/T256E (“YTE”), M428L/N434S (“LS”), orany combination thereof. Alternatively, or additionally, in anembodiment, the Fc region comprises (a) one or more (e.g., 2, 3, 4, 5,or all) combinations of mutations chosen from: T256D/Q311V/A378V,H285N/T307Q/N315D, H285D/T307Q/A378V, T307Q/Q311V/A378V,T256D/N286D/T307R/Q311V/A378V, or T256D/T307R/Q311V; (b) a mutation or acombination of mutations capable of disrupting an Fc effector function,e.g., L234A/L235A (also known as “LALA” mutation), or (c) both (a) and(b).

In an embodiment, the Fc region comprises mutations T256D/Q311V/A378Vand a mutation or a combination of mutations capable of disrupting an Fceffector function, e.g., L234A/L235A. In an embodiment, the Fc regioncomprises mutations H285N/T307Q/N315D and a mutation or a combination ofmutations capable of disrupting an Fc effector function, e.g.,L234A/L235A. In an embodiment, the Fc region comprises mutationsH285D/T307Q/A378V and a mutation or a combination of mutations capableof disrupting an Fc effector function, e.g., L234A/L235A. In anembodiment, the Fc region comprises mutations T307Q/Q311V/A378V and amutation or a combination of mutations capable of disrupting an Fceffector function, e.g., L234A/L235A. In an embodiment, the Fc regioncomprises mutations T256D/N286D/T307R/Q311V/A378V and a mutation or acombination of mutations capable of disrupting an Fc effector function,e.g., L234A/L235A. In an embodiment, the Fc region comprises mutationsT256D/T307R/Q311V and a mutation or a combination of mutations capableof disrupting an Fc effector function, e.g., L234A/L235A.

A reference Fc region amino acid sequence (including the numbering usedherein) is provided below (e.g., for identification of the mutationpositions described herein). The CH2 domain sequence is underlined; thehinge region sequence is in italics, and the CH3 domain sequence is inbold.

(SEQ ID NO: 1) 118ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 175QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA 232PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN 287AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQ P 344REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS 401DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Any of the half-life extension mutations described herein can be used incombination with any Fc mutation capable of enhancing or disrupting anFc effector function.

The Fc region can bind to various cell receptors (e.g., Fc receptors)and complement proteins. The Fc region can also mediate differentphysiological effects of antibody molecules, e.g., detection ofopsonized particles; cell lysis; degranulation of mast cells, basophils,and eosinophils; and other processes.

There are several different types of Fc receptors (FcR), which can beclassified based on the type of antibody that they recognize.

Fcγ receptors (FcγR) belong to the immunoglobulin superfamily, and areinvolved, e.g., in inducing phagocytosis of opsonized microbes. Thisfamily includes several members, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB(CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), which differ in theirantibody affinities due to their different molecular structure. Forinstance, FcγRI can bind to IgG more strongly than FcγRII or FcγRIIIdoes. FcγRI also has an extracellular portion comprising threeimmunoglobulin (Ig)-like domains, one more domain than FcγRII or FcγRIIIhas. This property allows FcγRI to bind a sole IgG molecule (ormonomer), but Fcγ receptors generally need to bind multiple IgGmolecules within an immune complex to be activated.

The Fcγ receptors differ in their affinity for IgG and the different IgGsubclasses can have unique affinities for each of the Fcγ receptors.These interactions can be further tuned by the glycan (oligosaccharide)at certain position of IgG. For example, by creating steric hindrance,fucose containing CH2-84.4 glycans reduce IgG affinity for FcγRIIIA,whereas GO glycans, which lack galactose and terminate instead withGlcNAc moieties, have increased affinity for FcγRIIIA (Maverakis et al.(2015) Journal of Autoimmunity 57 (6): 1-13)

The neonatal Fc receptor (FcRn) is expressed on multiple cell types andis similar in structure to MHC class I. This receptor also binds IgG andis involved in preservation of this antibody (Zhu et al. (2001). Journalof Immunology 166 (5): 3266-76.). FcRn is also involved in transferringIgG from a mother either via the placenta to her fetus or in milk to hersuckling infant. This receptor may also play a role in the homeostasisof IgG serum levels.

FcαRI (or CD89) belongs to the FcαR subgroup. FcαRI is found on thesurface of neutrophils, eosinophils, monocytes, macrophages (includingKupffer cells), and dendritic cells. It comprises two extracellularIg-like domains, and is a member of both the immunoglobulin superfamilyand the multi-chain immune recognition receptor (MIRR) family. Itsignals by associating with two FcRγ signaling chains.

Fc-alpha/mu receptor (Fcα/μR) is a type I transmembrane protein. It canbind IgA, although it has higher affinity for IgM (Shibuya and Honda(2006) Springer Seminars in Immunopathology 28 (4): 377-82). With oneIg-like domain in its extracellular portion, this Fc receptor is also amember of the immunoglobulin superfamily.

There are two known types of FcεR. The high-affinity receptor FcεRI is amember of the immunoglobulin superfamily (it has two Ig-like domains).FcεRI is found on epidermal Langerhans cells, eosinophils, mast cellsand basophils. This receptor can play a role in controlling allergicresponses. FcεRI is also expressed on antigen-presenting cells, andcontrols the production of immune mediators, e.g., cytokines thatpromote inflammation (von Bubnoff et al. (2003) Clinical andExperimental Dermatology 28 (2): 184-7). The low-affinity receptorFcεRII (CD23) is a C-type lectin. FcεRII has multiple functions as amembrane-bound or soluble receptor. It can also control B cell growthand differentiation and blocks IgE-binding of eosinophils, monocytes,and basophils (Kikutani et al. (1989) Ciba Foundation Symposium 147:23-31).

In an embodiment, the Fc region can be engineered to contain anantigen-binding site to generate an Fcab fragment (Wozniak-Knopp et al.(2010) Protein Eng Des 23 (4): 289-297). Fcab fragments can be insertedinto a full immunoglobulin by swapping the Fc region, thus obtaining abispecific antibody (with both Fab and Fcab regions containing distinctbinding sites).

The binding and recycling of FcRn can be illustrated below. For example,IgG and albumin are internalized into vascular endothelial cells throughpinocytosis. The pH of the endosome is 6.0, facilitating associationwith membrane-bound FcRn. The contents of endosomes can be processed inone of two ways: either recycling back to the apical cell membrane ortranscytosis from the apical to the basolateral side. IgG not associatedwith FcRn is degraded by lysosomes.

While not wishing to be bound by theory, it is believed that FcRninteraction with IgG is mediated through Fc. The binding of Fc to FcRnis pH specific, e.g., no significant binding at pH 7.4 and strongbinding in acidic environment. Structure of FcRn in complex with Fcdomain of IgG1 molecule is known in FIG. 1. Each FcRn molecule generallybinds to an Fc-monomer. In an embodiment, Fab domains can also influencebinding of IgG to FcRn, e.g., have either a negative or no influence onthe affinity of the IgG for FcRn.

There can be multiple considerations when an Fc region is engineered toenhance half-life of a polypeptide. For example, prolonging half-lifeand efficient recirculation of antibody molecules or fusion proteinsoften requires pH specific affinity enhancement (e.g., only at low pH ofthe endosome). FcRn binds proximal to the linker region between CH2 andCH3 domains of a Fc region. Modifications to the linker can impact Fcengagement with Fcγ receptors. Modifications on the Fc region can impactthermal stability and aggregation properties of the polypeptide.

FcRn Binding Optimization: Reducing Impact on Other Effector Functions

In an embodiment, the polypeptide (e.g., antibody molecule or fusionprotein) described herein has the same affinity function, or does notsubstantially alter (e.g., decrease by more than 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90%) an effector function (e.g., an effectorfunction described herein). In an embodiment, the effector function isnot associated with the binding between an Fc region and an FcRn.

The amino acid residues to be mutated can be selected, at least in part,based on the structural or functional properties of one or more bindingsites on the Fc region. These binding sites include, but are not limitedto, a Protein A binding site, a C1q binding site, an FcγRI binding site,an FcγRIIa binding site, an FcγRIIIa binding site, or an FcRn bindingsite, e.g., as shown in FIG. 2. The binding sites can also include aTRIM21 binding site, e.g., one or more residues chosen from loop308-316, loop 252-256, or loop 429-436 of an IgG. In an embodiment, thelinker region between the CH2 and CH3 domain can influence the dynamicsof the CH2 domain which impinges on FcγR binding.

Structural Basis for pH Specific Engagement of FcRn

Without wishing to be bound by theory, it is believed that low pH of theendosome leads to protonation of surface histidines on the CH2 and CH3domains. For example, protonation of residue H310 on CH2 and/or H433 onCH3 can be important for FcRn engagement, e.g., at low pH (e.g., at pH6.0). Protonation can also lead to change in the conformational dynamicsof the region, such as better exposure or shielding of the linker regionfor solvent or ligand molecule binding. Accordingly, in an embodiment,the polypeptide (e.g., antibody molecule or fusion protein) comprises amutation in residue H310, a mutation in residue H433, or both. One ormore residues adjacent to residues H310 and/or H433 can also be mutated.The polypeptide can also include a compensating or beneficial mutation,e.g., a mutation that compensates, or beneficial, for any of theaforesaid mutations, e.g., to reduce a negative consequence of thatmutation (e.g., polar vs. non-polar, charged vs. no charge,positively-charged (basic) vs. negatively charged (acidic), orhydrophobic vs. hydrophilic). For example, P247D can be a compensatingor beneficial mutation.

In an embodiment, protonation of histidine can result in additionalconformational changes including, e.g., movement/displacement of thelinker/CH2/CH3 interface residues. Crystal structures of Fc fragmentshave been crystallized at different pHs. As shown in FIG. 3, an analysisof two high resolution crystal structures of Fc fragments crystallizedand pH 6.5 (cyan) and 5.0 (green) indicated potential differences.

Mapping of Fc-FcRn Interaction Interface

In an embodiment, the polypeptide (e.g., antibody molecule or fusionprotein) described herein comprises a mutation that can alter theinteraction between an Fc region and an FcRn. In an embodiment, themutation is selected based, at least in part, on a structural feature ofthe Fc-FcRn interaction interface.

In an embodiment, the Fc region of an immunoglobulin monomer can havethe structure shown in FIG. 4. As shown in FIG. 4, the black dotted lineindicates the Fc-FcRn interaction interface. The structure includes FcRncontact residues and FcRn affinity enhancing Fc residues, e.g., asdescribed herein. Residues H310 and H435, which are located in the CH2and CH3 domains, respectively, are primarily responsible for the pHdependent Fc-FcRn interactions. In an embodiment, the FcRn binds the Fcregion between its CH2 and CH3 domains. In another embodiment, theFc-FcRn binding site is located across both the CH2 and CH3 domains ofthe Fc monomer.

Network View of Fc-FcRn Engagement Provides Insights on Co-Substitutionsof Amino Acids

In an embodiment, the polypeptide (e.g., antibody molecule or fusionprotein) described herein comprises a plurality of mutations that canalter the interaction between an Fc region and an FcRn. In anembodiment, the mutation is selected based, at least in part, on anetwork view of the Fc-FcRn interaction.

Without wishing to be bound by theory, it is believed that in anembodiment, residue H310 plays a central role in engagement with theFcRn, e.g., as determined by a network analysis of the Fc-FcRn complex.As shown in FIG. 5, residue H310 is highly interconnected to multipleother highly networked residues. In an embodiment, a mutation in theH310 cluster, and neighboring (connected nodes), can strengthen the H310network. Analysis of sub-networks informs introduction of synergisticmutations for favorable FcRn interaction; with reduced or minimal impacton other Fc residues.

Design Considerations for Optimizing FcRn Binding

In an embodiment, the polypeptide (e.g., antibody molecule or fusionprotein) described herein can be designed for optimizing Fc-FcRnbinding.

In an embodiment, the polypeptide having a mutation in the Fc region hasa pH-specific affinity enhancement, compared to a reference polypeptide(e.g., an otherwise identical polypeptide without the mutation). In anembodiment, affinity enhancement is achieved by increasing van der Waalinteraction. In an embodiment, affinity enhancement is not achieved byintroduction of hydrogen bonds and/or electrostatic interaction. In anembodiment, the mutation does not alter, or has reduced or minimalperturbation to, the conformation of the linker region between the CH2and CH3 domains. In an embodiment, the polypeptide comprises a pluralityof mutations across both domains (four quadrants). In an embodiment, thepolypeptide does not contain a large cluster of hydrophobic or aromaticresidues on the surface.

In an embodiment, the polypeptide comprises a mutation that enhances thestrength of interaction between an Fc region and FcRn or reduces thedissociation constant (K_(d)) for FcRn, e.g., at an acidic pH. In anembodiment, the polypeptide comprises a mutation that reduces the rateof dissociation (k_(off)) for FcRn, e.g., at an acidic pH. In anembodiment, the polypeptide comprises a mutation that increases the rateof association (k_(on)) for FcRn, e.g., at an acidic pH. In anembodiment, the polypeptide comprises a mutation that reduces the rateof dissociation (k_(off)) for FcRn, and increases the rate ofassociation (k_(on)) for FcRn, e.g., at an acidic pH. In an embodiment,the polypeptide comprises a mutation that reduces the rate ofdissociation (k_(off)) for FcRn, and does not, or does notsignificantly, affect the rate of association (k_(on)) for FcRn, e.g.,at an acidic pH. Without wishing to be bound by theory, it is believedthat in an embodiment, the reduction of the dissociation constant K_(d)for FcRn is primarily resulted from the reduction of the rate ofdissociation (k_(off)) for FcRn, rather than the increase of the rate ofassociation (k_(on)).

Experimental Evaluation: Fc Mutations Assessed Along Multiple Dimensionsand by Different Assay Platforms

The polypeptides (e.g., antibody molecules or fusion proteins) describedherein can be evaluated by a number of methods. The pH-specific Fc-FcRnbinding (e.g., binding at pH6.0 and pH7.4) can be determined, e.g., byan Octet-based assay, a competition assay (e.g., flow cytometry), orsurface plasmon resonance (SPR).

Biophysical characterization (e.g., thermal stability, aggregation, orexpression) can be performed. Thermal stability can be determined, e.g.,by SYPRO orange. Aggregation can be measured, e.g., by SEC or RP-HPLC.

Effector functions, e.g., relating to FcγRI (e.g., by an Octet-basedassay), FcγRIIIa, FcγRIIa, FcγRIIb (e.g., by ELISA), C1q, ADCC, or CDC,can be tested.

Tripartite Motif-Containing Protein 21 (TRIM21) binding can be tested.TRIM21 is a cytosolic receptor that binds with Fc of IgG. TRIM21 plays arole in mediating intracellular recognition and neutralization of Fcbound pathogens such as viruses, bacteria and fungus. For example,TRIM21 plays an important role in the neutralization of non-envelopedviruses (McEwan et al. Nat Immunol. 2013; 14(4):327-36). Its role hasbeen further expanded to include directing of immune complexes fordegradation (McEwan et al. Proc Natl Acad Sci USA. 2017;114(3):574-579). TRIM21 binds to the CH2:CH3 interface of the antibodyFc region, which overlaps with the FcRn binding site. Some Fc mutationsthat increase FcRn affinity decrease TRIM21 affinity (Foss et al. JImmunol. 2016; 196(8):3452-3459). Key contact residues include, e.g.,positions 253, 433, 434, and 435. The LS variant (M428L/N434S) containsa mutation at position 434, and it has been shown that the N434Smutation causes a 10 fold decrease in TRIM21 binding. TRIM21-mediatedneutralization is known as antibody dependent intracellularneutralization (ADIN).

Mucosal uptake can be tested. FcRn transports IgG across differentcellular barriers such as the mucosal epithelium lining the intestineand the alveolar surfaces. Modification of FcRn binding provides amechanism to enhance mucosal localization that confers immuneprotection.

The half-life of the polypeptide can be measured, e.g., using transgenicmice (e.g., Tg32 and Tg276 mice from Jackson's lab), or in primates(e.g., cynomolgus monkeys).

Exemplary assays are described in more detail as follows:

1. FcRn Binding Assays

a. Octet Assay with Immobilization of FcRn to NiNTA Biosensors

Immobilization of FcRn to a NiNTA biosensor via a 6× histidine tag (SEQID NO: 2) allows for subsequent interrogation of binding to IgGmolecules under acidic (pH 6.0) and physiological (pH 7.4) conditions.This strategy has been previously described (1) and this method detailsan adaptation of the referenced protocol. Briefly, recombinant humanFcRn at 5 μg/mL is loaded onto a NiNTA biosensor for 180 seconds. Aftera 60 second baseline step in 1×PBS pH 6.0, the FcRn loaded tip isexposed to IgG at a concentration of 250 nM (37.5 μg/mL) for 60 seconds,followed by dissociation for 60 seconds in PBS pH 6.0, and an additional30 seconds in PBS pH 7.4. After assay completion, a quantitativeassessment of the affinity constant (K_(D)) at pH 6.0 is performed usingthe ForteBio octet software and a qualitative assessment is performed byplotting the response rate over time, allowing for visualization of theassociation of IgG to FcRn at pH 6.0 and the subsequent dissociation atpH 6.0 and pH 7.4.

b. Octet Assay with Immobilization of IgG to Anti-CH1 Biosensors

Immobilization of IgG to an anti-CH1 biosensor allows for subsequentinterrogation of binding to FcRn molecules under acidic (pH 6.0) andphysiological (pH 7.4) conditions. This strategy has been previouslydescribed (2) and this method details an adaptation of the referencedprotocol. Briefly, purified IgG at 5 μg/mL is loaded onto an anti-CH1biosensor for 180 seconds. After a 60 second baseline step in 1×PBS pH6.0, the IgG loaded tip is exposed to FcRn at a concentration of 50μg/mL for 60 seconds, followed by dissociation for 60 seconds in PBS pH6.0, and an additional 30 seconds in PBS pH 7.4. After assay completion,a quantitative assessment of the affinity constant (K_(D)) at pH 6.0 isperformed using the ForteBio octet software and a qualitative assessmentis performed by plotting the response rate over time, allowing forvisualization of the association of IgG to FcRn at pH 6.0 and thesubsequent dissociation at pH 6.0 and pH 7.4.

c. Cell Based Assay

Cell-based assays are also used to analyze the interactions between FcRnand IgG (Ref: PMID: 23384837). Expression of membrane-anchored FcRn onthe cell surface closely represents the physiological presentation ofFcRn, where the plasma membrane and molecular orientations influenceinteractions between FcRn and IgG. The assay used here is a competitionassay in which IgGs of interest are evaluated for their ability tocompete with cell binding of a fluorescently-labeled, high affinity Fccompetitor reagent (Fc-A488). Expi293 cells expressing the full-length,membrane-bound FcRn heterodimer are incubated with mixtures of a staticconcentration of Fc-A488 (0.5 ug/ml) and varying concentrations of IgGof interest (0.001-10 μM). Cell-bound fluorescence is detected by flowcytometry. IgGs with improved binding to FcRn will compete off the Fccompetitor at lower IgG concentrations. This assay is robust, linear,and specific and can be used to show differences in relative binding ofIgG/Fc variants to FcRn.

2. Thermal Stability Assay

The stability of the IgG variants was assessed by Differential ScanningFluorimetry (DSF) is an assay using SYPRO® Orange dye to monitor proteinunfolding under thermal stress. SYPRO® Orange is a fluorescent dye thatnon-specifically binds to hydrophobic surfaces and its fluorescence isquenched in aqueous environments. Proteins begin to lose their secondarystructure and unfold with increasing temperatures thus exposing theirhydrophobic core residues, and allowing the dye to fluoresce. Maximumfluorescent signal is attained at complete unfolding, after which theprotein begins to aggregate, reducing the exposed hydrophobic residues,and thus reducing the fluorescent signal. At the midpoint between nativestate and fully unfolded protein is the transition temperature, or melttemperature (Tm), which can be used to directly compare proteinconstructs or formulation conditions for relative stability.

In an embodiment, factors other than FcRn binding can also affect theobserved half-life of a polypeptide (e.g., an antibody molecule orfusion protein). These factors can include, e.g., aggregationpropensity, non-specific binding, stability, or Fab composition.

In an embodiment, a mutation in a non-Fc region is introduced, e.g., toprovide a substantial improvement in half-life in the context of asuboptimal template. For example, polypeptides (e.g., antibody moleculesor fusion proteins) with lower starting half-life may indicate thepresence of suboptimal properties. Engineering efforts can also focus onmitigating the root cause of lower half-life, e.g., to maintainrequisite binding profile (e.g., affinity and/or specificity), or toobtain suitable developability characteristics (e.g., stability,solubility, expression level, or aggregation). In an embodiment,additional engineering is performed for polypeptide (e.g., antibodymolecules or fusion proteins) with suboptimal biophysical properties torealize maximal half-life extension.

Pharmacokinetics

The polypeptides (e.g., antibody molecules or fusion proteins) describedherein can have one or more desired pharmacokinetic properties, e.g.,one or more (e.g., 2, 3, 4, 5, or more) of the pharmacokineticproperties described herein.

Pharmacokinetics (PK) can be used to determine the fate of substancesadministered to a living organism. PK studies can be used to analyzedrug metabolism and to identify the fate of a drug from the moment thatit is administered up to the point at which it is completely eliminatedfrom the body. Pharmacokinetics describes, e.g., how the body affects aspecific drug after administration through the mechanisms of absorptionand distribution, the chemical changes of the substance in the body(e.g. by metabolic enzymes such as cytochrome P450 orglucuronosyltransferase enzymes), or the effects and routes of excretionof the metabolites of the drug. Pharmacokinetic properties of drugs maybe affected by elements such as the site of administration and the doseof administered drug, which may affect the absorption rate.Pharmacokinetics can be analyzed in conjunction with pharmacodynamics(e.g., the study of the biochemical and physiologic effects of drugs).

A number of different models have been developed for pharmacokinetics.For example, pharmacokinetic modelling can be performed bynoncompartmental or compartmental methods.

Noncompartmental methods estimate the exposure to a drug by estimatingthe area under the curve of a concentration-time graph. NoncompartmentalPK analysis is highly dependent on estimation of total drug exposure.Total drug exposure is often estimated by area under the curve (AUC)methods, with the trapezoidal rule (numerical integration) the mostcommon method. Due to the dependence on the length of ‘x’ in thetrapezoidal rule, the area estimation is highly dependent on theblood/plasma sampling schedule. That is, the closer time points are, thecloser the trapezoids reflect the actual shape of the concentration-timecurve.

Compartmental methods estimate the concentration-time graph usingkinetic models. Compartmental PK analysis uses kinetic models todescribe and predict the concentration-time curve. PK compartmentalmodels are often similar to kinetic models used in other scientificdisciplines such as chemical kinetics and thermodynamics. The advantageof compartmental over some noncompartmental analyses is the ability topredict the concentration at any time. Compartment-free modelling basedon curve stripping does not suffer this limitation. The simplest PKcompartmental model is the one-compartmental PK model with IV bolusadministration and first-order elimination. The more complex PK models(e.g., PBPK models) rely on the use of physiological information to easedevelopment and validation.

In a single-compartment model, the graph of the relationship between thevarious factors involved (e.g., dose, blood plasma concentrations, orelimination) gives a straight line or an approximation to one (i.e.,linear pharmacokinetics). For drugs to be effective they need to be ableto move rapidly from blood plasma to other body fluids and tissues.

In multi-compartmental models, the graph for the non-linear relationshipbetween the various factors is represented by a curve, the relationshipsbetween the factors can then be found by calculating the dimensions ofdifferent areas under the curve. The models used in non-linearpharmacokinetics are largely based on Michaelis-Menten kinetics.

The various compartments that the model is divided into are commonlyreferred to as the ADME scheme (also referred to as LADME if liberationis included as a separate step from absorption): liberation (e.g., theprocess of release of a drug from the pharmaceutical formulation);absorption (e.g., the process of a substance entering the bloodcirculation); distribution (e.g., the dispersion or dissemination ofsubstances throughout the fluids and tissues of the body); metabolism(or biotransformation, or inactivation) (e.g., the recognition by theorganism that a foreign substance is present and the irreversibletransformation of parent compounds into daughter metabolites); andexcretion (e.g., the removal of the substances from the body). In rarecases, some drugs irreversibly accumulate in body tissue. The two phasesof metabolism and excretion can also be grouped together under the titleelimination.

All these parameters can be represented through mathematical formulasthat have a corresponding graphical representation. The use of thesemodels allows an understanding of the characteristics of a molecule, aswell as how a particular drug will behave given information regardingsome of its basic characteristics such as its acid dissociation constant(pKd), bioavailability and solubility, absorption capacity anddistribution in the organism.

The model outputs for a drug can be used in industry (for example, incalculating bioequivalence when designing generic drugs) or in theclinical application of pharmacokinetic concepts. Clinicalpharmacokinetics provides a number of performance guidelines foreffective and efficient use of drugs for human-health professionals andin veterinary medicine.

Exemplary pharmacokinetic properties include, but are not limited to,dose (e.g., the amount of drug administered), dosing interval (e.g., thetime between drug dose administrations), C_(max) (e.g., the peak plasmaconcentration of a drug after administration), t_(max) (e.g., the timeto reach C_(max)), C_(min) (e.g., the lowest concentration that a drugreaches before the next dose is administered), volume of distribution(e.g., the apparent volume in which a drug is distributed, e.g.,relating drug concentration to drug amount in the body), concentration(e.g., the amount of drug in a given volume of plasma), half-life orelimination half-life (e.g., the time required for the concentration ofthe drug to reach half of its original value), elimination rate constant(e.g., the rate at which a drug is removed from the body), infusion rate(e.g., the rate of infusion required to balance elimination), area underthe curve (e.g., the integral of the concentration-time curve, e.g.,after a single dose or in steady state), clearance (e.g., the volume ofplasma cleared of the drug per unit time), bioavailability (e.g., thesystemically available fraction of a drug), or fluctuation (e.g., thepeak trough fluctuation within one dosing interval at steady state).

Pharmacokinetic properties can be measured by various methods. Forexample, bioanalytical methods can be used to construct aconcentration-time profile. Chemical techniques can be employed tomeasure the concentration of drugs in biological matrix, e.g., plasma.Proper bioanalytical methods should be selective and sensitive. Forexample, microscale thermophoresis can be used to quantify how thebiological matrix/liquid affects the affinity of a drug to its target(Baaske et al. (2010). Angew. Chem. Int. Ed. 49 (12): 1-5; Wienken etal. (2010). Nature Communications 1 (7): 100).

Pharmacokinetic properties can also be studied using mass spectrometry,e.g., when there is a need for high sensitivity to observeconcentrations after a low dose and a long time period. A commoninstrumentation used in this application is LC-MS with a triplequadrupole mass spectrometer. Tandem mass spectrometry can be employedfor added specificity. Standard curves and internal standards can beused for quantitation of a pharmaceutical in the samples. The samplesrepresent different time points as a pharmaceutical is administered andthen metabolized or cleared from the body. Blank samples taken beforeadministration are used in determining background and ensuring dataintegrity with such complex sample matrices. The standard curve can belinear, or curve fitting can be used with more complex functions such asquadratics since the response of most mass spectrometers is less thanlinear across large concentration ranges (Hsieh and Korfmacher (2006)Current Drug Metabolism 7 (5): 479-89; Covey et al. (1986) Anal. Chem.58 (12): 2453-60; Covey et al. (1985). Anal. Chem. 57 (2): 474-81).

Exemplary Antibody Molecules

The methods described herein can be used to engineer a variety ofantibody molecules, e.g., any antibody molecule containing an Fc region.

In an embodiment, the antibody molecule is a chimeric antibody molecule,a humanized antibody molecule, or a human antibody molecule.

In an embodiment, the antibody molecule is a whole monoclonal antibody.In another embodiment, the antibody molecule is an Fc region-containingderivative of an antibody molecule that does not contain an Fc region(e.g., an antigen-binding fragment described herein). Exemplaryantigen-binding fragments include, but are not limited to, Fab, F(ab′)2,Fab′, scFv, di-scFv, or sdAb. In another embodiment, the antibodymolecule is a bispecific monoclonal antibody, e.g., a trifunctionalantibody (3funct) or bi-specific T-cell engager (BiTE).

In an embodiment, the antibody molecule targets a molecule (e.g., aprotein) associated with an infectious disease (e.g., a viral infection,a bacterial infection, or a fungal infection). In another embodiment,the antibody molecule targets a molecule (e.g., a protein) or cellassociated with a cancer. In another embodiment, the antibody moleculetargets a molecule (e.g., a protein) or cell associated with an immunedisorder. In another embodiment, the antibody molecule targets amolecule (e.g., a protein) or cell associated with a cardiovasculardisorder. In another embodiment, the antibody molecule targets amolecule (e.g., a protein) or cell associated with a metabolic disorder.In another embodiment, the antibody molecule targets a molecule (e.g., aprotein) or cell associated with a neurological disorder.

Exemplary antibody molecules include, but are not limited to, antibodymolecules that target one or more (e.g., 2) of the following moleculesor cells: β-amyloid, 4-1BB, SAC, 5T4, ACF9, ACFIX, activin receptor-likekinase 1, ACVR2B, an adenocarcinoma antigen, AGS-22M6,alpha-fetoprotein, angiopoietin 2, angiopoietin 3, a protective antigenof anthrax toxin, AOC3 (VAP-1), B7-H3, bacillus anthracisanthrax, BAFF,B-lymphoma cell, C242 antigen, C5, CA-125 (imitation), calcitonin, canislupus familiaris IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin,CCL11 (eotaxin-1), CCR2, CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147(basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22,CD221, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD27,CD274, CD276, CD28, CD3, CD3 epsilon, CD30 (TNFRSF8), CD33, CD37, CD38(cyclic ADP ribose hydrolase), CD4, CD40, CD40 ligand, CD41 (integrinalpha-IIb), CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B,CD80, CEA, a CEA-related antigen, CFD, CGRP, ch4D5, CLDN18.2,Clostridium difficile, clumping factor A, coagulation factor III, CSF1R,CSF2, CTGF, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus,cytomegalovirus glycoprotein B, dabigatran, DLL3, DLL4, DPP4, DRS, E.coli shiga toxintype-1, E. coli shiga toxintype-2, EGFL7, EGFR,endoglin, endotoxin, EpCAM, ephrin receptor A3, episialin, ERBB3,Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrinII beta chain, fibronectin extra domain-B, folate hydrolase, folatereceptor 1, folate receptor alpha, Frizzled receptor, ganglioside GD2,GCGR, GD2, GD3 ganglioside, GDF-8, glypican 3, GMCSF receptor α-chain,GPNMB, growth differentiation factor8, GUCY2C, hemagglutinin, hepatitisB surface antigen, hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR,histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factorreceptor kinase, human TNF, human beta-amyloid, ICAM-1 (CD54), ICOSL,IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-I, IGHE, IL 20,IL-1, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL2, IL-22, IL-23,IL23A, IL31RA, IL-4, IL-4, IL-5, IL6, IL-6 receptor (IL6R), IL-9, ILGF2,influenza A virus hemagglutinin (HA), insulin-like growth factor Ireceptor, integrin α4, integrin α4β7, integrin α5β1, integrin α7β7,integrin αIIbβ3, integrin αvβ3, interferon receptor, interferon α/βreceptor, interferon gamma-induced protein, ITGA2, ITGB2 (CD18),kallikrein, KIR2D, KLRC1, Lewis-Y antigen, LFA-1 (CD11a), LFA-1 (CD11a),LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1,mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associatedglycoprotein, myostatin, NCA-90 (granulocyte antigen), NCA-90(granulocyte antigen), neural apoptosis-regulated proteinase 1, neuralapoptosis-regulated proteinase 1, NGF, NGF, N-glycolylneuraminic acid,NOGO-A, Notch 1, Notch receptor, NRP1, Oryctolagus cuniculus, OX-40,oxLDL, PCSK9, PD-1, PD-1, PDCD1, PDGF-R α, phosphate-sodiumco-transporter, phosphatidylserine, platelet-derived growth factorreceptor beta, prostatic carcinoma cells, Pseudomonas aeruginosa,Pseudomonas aeruginosa type III secretion system, rabies virusglycoprotein, rabies virus glycoprotein, RANKL, respiratory syncytialvirus, respiratory syncytial virus, RHD, Rhesus factor, Rhesus factor,RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST,sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T-cellreceptor, TEM1, tenascin C, TFPI, TGF beta 1, TGF beta 2, TGF-β, TNFRsuperfamily member 4, TNF-α, TRAIL-R1, TRAIL-R2, TSLP, tumor antigenCTAA16.88, tumor specific glycosylation of MUC1, tumor-associatedcalcium signal transducer 2, TWEAK receptor, TYRP1(glycoprotein 75),VEGFA, VEGFR-1, VEGFR2, vimentin, or VWF.

Exemplary antibody molecules include, but are not limited to, antibodymolecules that target one or more (e.g., 2) of the following pathogens(e.g., bacteria, viruses, or fungi): Actinomyces gerencseriae,Actinomyces israelii, Actinomycetoma species, Alphavirus, Anaplasmaphagocytophilum, Anaplasma species, Ancylostoma braziliense, Ancylostomaduodenale, Angiostrongylus, Anisakis, Arcanobacterium haemolyticum,Ascaris lumbricoides, Aspergillus species, Astroviridae family, Babesiaspecies, Bacillus anthracis, Bacillus cereus, bacterial vaginosismicrobiota, Bacteroides species, Balantidium coli, Bartonella,Bartonella bacilliformis, Bartonella henselae, Batrachochytriumdendrabatidis, Baylisascaris species, BK virus, Blastocystis species,Blastomyces dermatitidis, Bordetella pertussis, Borrelia afzelii,Borrelia burgdorferi, Borrelia garinii, Borrelia hermsii, Borreliarecurrentis, Borrelia species, Brucella species, Brugia malayi,Bunyaviridae family, Burkholderia cepacia, Burkholderia mallei,Burkholderia pseudomallei, Burkholderia species, Caliciviridae family,Campylobacter species, Candida albicans, Candida species, Capillariaaerophila, Capillaria hepatica, Capillaria philippinensis, Chlamydiatrachomatis, Chlamydia trachomatis, Chlamydia trachomatis, Chlamydophilapneumoniae, Chlamydophila psittaci, Clonorchis sinensis, Clostridiumbotulinum, Clostridium difficile, Clostridium perfringens, Clostridiumperfringens, Clostridium tetani, Clostridium species, Coccidioidesimmitis, Coccidioides immitis, Coccidioides posadasii, Coccidioidesposadasii, Colorado tick fever virus (CTFV), coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Coxsackie A virus andEnterovirus 71 (EV71), Crimean-Congo hemorrhagic fever virus,Cryptococcus neoformans, Cryptosporidium species, Cyclosporacayetanensis, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 orDEN-4), Dientamoeba fragilis, Diphyllobothrium, Dracunculus medinensis,Ebolavirus (EBOV), Echinococcus species, Ehrlichia chaffeensis,Ehrlichia ewingii, Ehrlichia species, Entamoeba histolytica,Enterobacteriaceae family, Enterobius vermicularis, Enterococcusspecies, Enterovirus species, Enteroviruses, Entomophthorales order(Entomophthoramycosis), Epidermophyton floccosum, Epstein-Barr Virus(EBV), Escherichia coli O157:H7, Eumycetoma species, Fasciola hepaticaand Fasciola gigantica, Fasciolopsis buski, Filarioidea superfamily,Flavivirus, Fonsecaea pedrosoi, Francisella tularensis, Fusobacteriumspecies, Geotrichum candidum, Giardia lamblia, Gnathostoma hispidum,Gnathostoma spinigerum, Green algae Desmodesmus armatus, Group AStreptococcus, Guanarito virus, Haemophilus ducreyi, Haemophilusinfluenzae, Heartland virus, Helicobacter pylori, Hepatitis A virus,Hepatitis B virus, Hepatitis C virus, Hepatitis D Virus, Hepatitis Evirus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasmacapsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Humanbocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7(HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Hymenolepis diminuta, Hymenolepis nana,Isospora belli, JC virus, Junin virus, Kingella kingae, Klebsiellagranulomatis, Lassa virus, Legionella pneumophila, Leishmania species,Leptospira species, Listeria monocytogenes, Lymphocytic choriomeningitisvirus (LCMV), Machupo virus, Malassezia species, Marburg virus, Measlesvirus, Measles virus, Metagonimus yokagawai, Microsporidia phylum,Middle East respiratory syndrome coronavirus, Molluscum contagiosumvirus (MCV), Monkeypox virus, Mucorales order (Mucormycosis), Mumpsvirus, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacteriumtuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleriafowleri, Necator americanus, Neisseria gonorrhoeae, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia asteroids, Nocardiaspecies, O111 and O104:H4, Onchocerca volvulus, Opisthorchis felineus,Opisthorchis viverrini, Orthomyxoviridae family, Paracoccidioidesbrasiliensis, Paragonimus westermani, Paragonimus species, parasiticdipterous fly larvae, Parvovirus B19, Pasteurella species, Pediculushumanus capitis, Pediculus humanus corporis, Phthirus pubis, Piedraiahortae, Plasmodium species, Pneumocystis jirovecii, Poliovirus,Prevotella species, PRNP, Propionibacterium propionicus, Rabies virus,Respiratory syncytial virus (RSV), Rhinosporidium seeberi, Rhinovirus,rhinoviruses, Rickettsia, Rickettsia akari, Rickettsia prowazekii,Rickettsia rickettsii, Rickettsia typhi, Rickettsia species, Rift Valleyfever virus, Rotavirus, Rubella virus, Sabia, Salmonella enterica subsp.enterica, Salmonella species, Sarcoptes scabiei, SARS coronavirus,Schistosoma species, serovar typhi, Shigella species, Sin Nombre virus,Sporothrix schenckii, Staphylococcus, Staphylococcus species,Staphylococcus species, Streptococcus agalactiae, Streptococcuspneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taeniasolium, Taenia species, Toxocara canis, Toxocara cati, Toxoplasmagondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis,Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton rubrum,Trichophyton tonsurans, Trichophyton species, Trichosporon beigelii,Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasmaurealyticum, Varicella zoster virus (VZV), Variola major, Variola minor,Venezuelan equine encephalitis virus, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificus, West Nile virus, Wuchereriabancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, or Zika virus,

Exemplary antibody molecules include, but are not limited to, 3f8, 8h9,abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab,adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab,alacizumab pegol, a1d518, alemtuzumab, alirocumab, altumomab pentetate,amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab,anrukinzumab (ima-638), apolizumab, arcitumomab, ascrinvacumab,aselizumab, atezolizumab, atinumab, atlizumab (tocilizumab),atorolimumab, avelumab, bapineuzumab, basiliximab, bavituximab,bectumomab, begelomab, belimumab, benralizumab, bertilimumab,besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab,bimekizumab, bivatuzumab mertansine, bleselumab, blinatumomab,blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab vedotin,briakinumab, brodalumab, brolucizumab, brontictuzumab, cabiralizumab,canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab,capromab pendetide, carlumab, carotuximab, catumaxomab,cbr96-doxorubicin immunoconjugate, cedelizumab, cergutuzumabamunaleukin, certolizumab pegol, cetuximab, ch.14.18, citatuzumabbogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumabtetraxetan, codrituzumab, coltuximab ravtansine, conatumumab,concizumab, crenezumab, crotedumab, cr6261, dacetuzumab, daclizumab,dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab,denintuzumab mafodotin, denosumab, derlotuximab biotin, detumomab,dinutuximab, diridavumab, domagrozumab, dorlimomab aritox, drozitumab,duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab,eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab,elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab,emicizumab, enavatuzumab, enfortumab vedotin, enlimomab pegol,enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomabcituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab,etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab,faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab,fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab,fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab,fulranumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab,gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab,glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab,ibritumomab tiuxetan, icrucumab, idarucizumab, igovomab, imab362,imalumab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine,indusatumab vedotin, inebilizumab, infliximab, intetumumab, inolimomab,inotuzumab ozogamicin, ipilimumab, iratumumab, isatuximab, itolizumab,ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab,lanadelumab, landogrozumab, laprituximab emtansine, lebrikizumab,lemalesomab, lendalizumab, lenzilumab, lerdelimumab, lexatumumab,libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan,lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumabmertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab,mapatumumab, margetuximab, maslimomab, mavrilimumab, matuzumab,mepolizumab, metelimumab, milatuzumab, minretumomab, mirvetuximabsoravtansine, mitumomab, mogamulizumab, monalizumab, morolimumab,motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox,namilumab, naptumomab estafenatox, naratuximab emtansine, narnatumab,natalizumab, navicixizumab, navivumab, nebacumab, necitumumab,nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab,nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab,ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab,onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab,orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab,ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab,pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab,pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab,pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab,placulumab, plozalizumab, pogalizumab, polatuzumab vedotin, ponezumab,prezalizumab, priliximab, pritoxaximab, pritumumab, pro 140, quilizumab,racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab,ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab,rilotumumab, rinucumab, risankizumab, rituximab, rivabazumab pegol,robatumumab, roledumab, romosozumab, rontalizumab, rovalpituzumabtesirine, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab,sapelizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab,setoxaximab, sevirumab, sibrotuzumab, sgn-cd19a, sgn-cd33a, sifalimumab,siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin,solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab,suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab,tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tefibazumab,telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab,tesidolumab, tetulomab, tezepelumab, tgn1412, ticilimumab(tremelimumab), tildrakizumab, tigatuzumab, timolumab, tisotumabvedotin, tnx-650, tocilizumab (atlizumab), toralizumab, tosatoxumab,tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumabemtansine, trbs07, tregalizumab, tremelimumab, trevogrumab, tucotuzumabcelmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab,ustekinumab, utomilumab, vadastuximab talirine, vandortuzumab vedotin,vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab,vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab,vobarilizumab, volociximab, vorsetuzumab mafodotin, votumumab,xentuzumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomabaritox, or derivative thereof.

In an embodiment, the antibody molecule comprises one, two, or threeCDRs of the VH region of an antibody molecule described herein, usingthe Kabat or Chothia definitions of CDRs. In an embodiment, the antibodymolecule comprises one, two, or three CDRs of the VL region of anantibody molecule described herein, using the Kabat or Chothiadefinitions of CDRs. In an embodiment, the antibody molecule comprisesone or more (e.g., two or three) CDRs of the VH region and/or one ormore (e.g., two or three) CDRs of the VL region of an antibody moleculedescribed herein, using the Kabat or Chothia definitions of CDRs.

In an embodiment, the antibody molecule comprises one, two, three, orfour frameworks of the VH region of an antibody molecule describedherein. In an embodiment, the antibody molecule comprises one, two,three, or four frameworks of the VL region of an antibody moleculedescribed herein. In an embodiment, the antibody molecule comprises oneor more (e.g., two, three, or four) frameworks of the VH region and/orone or more (e.g., two, three, or four) frameworks of the VL region ofan antibody molecule described herein.

In an embodiment, the antibody molecule comprises a heavy chain variableregion of an antibody molecule described herein, or a heavy chainvariable region having an amino acid sequence substantially identicalthereto (e.g., an amino acid sequence at least about 85%, 90%, 95%, 99%or more identical thereto, or which differs by no more than 1, 2, 5, 10,or 15 amino acid residues). In an embodiment, the antibody moleculecomprises a light chain variable region of an antibody moleculedescribed herein, or a light chain variable region having an amino acidsequence substantially identical thereto (e.g., an amino acid sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, or whichdiffers by no more than 1, 2, 5, 10, or 15 amino acid residues). In anembodiment, the antibody molecule comprises a heavy chain variableregion, or a heavy chain variable region having an amino acid sequencesubstantially identical thereto (e.g., an amino acid sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, or which differs byno more than 1, 2, 5, 10, or 15 amino acid residues), and a light chainvariable region of an antibody molecule described herein, or a lightchain variable region having an amino acid sequence substantiallyidentical thereto (e.g., an amino acid sequence at least about 85%, 90%,95%, 99% or more identical thereto, or which differs by no more than 1,2, 5, 10, or 15 amino acid residues).

In an embodiment, the antibody molecule further comprises a heavy chainconstant region, e.g., a heavy chain constant region of an antibodymolecule described herein, or a heavy chain constant region having anamino acid sequence substantially identical thereto (e.g., an amino acidsequence at least about 85%, 90%, 95%, 99% or more identical thereto, orwhich differs by no more than 1, 2, 5, 10, or 15 amino acid residues).In an embodiment, the antibody molecule further comprises a light chainconstant region, e.g., a light chain constant region of an antibodymolecule described herein, or a light chain constant region having anamino acid sequence substantially identical thereto (e.g., an amino acidsequence at least about 85%, 90%, 95%, 99% or more identical thereto, orwhich differs by no more than 1, 2, 5, 10, or 15 amino acid residues).In an embodiment, the antibody molecule further comprises a heavy chainconstant region, or a heavy chain constant region having an amino acidsequence substantially identical thereto (e.g., an amino acid sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, or whichdiffers by no more than 1, 2, 5, 10, or 15 amino acid residues), and alight chain constant region, or a light chain constant region having anamino acid sequence substantially identical thereto (e.g., an amino acidsequence at least about 85%, 90%, 95%, 99% or more identical thereto, orwhich differs by no more than 1, 2, 5, 10, or 15 amino acid residues).In an embodiment, the antibody molecule comprises a heavy chain constantregion, a light chain constant region, and heavy and light chainvariable regions of an antibody molecule, as described herein.

The antibody molecules described herein can have several advantageousproperties, including, but limited to, a desired (e.g., increased)half-life. For example, the antibody molecules can be used toeffectively treat, prevent or diagnose a disorder described herein.

In an embodiment, the antibody molecule is capable of binding to atarget molecule or cell. For example, the engineered antibody moleculeis capable of binding to the target molecule or cell, with the same, orsubstantially the same, binding specificity and/or affinity, as comparedto the parental antibody molecule. In an embodiment, the antibodymolecule binds to the target molecule or cell with high affinity, e.g.,with a dissociation constant (K_(d)) of less than about 100 nM,typically about 10 nM, and more typically, about 10-0.01 nM, about5-0.01 nM, about 3-0.05 nM, about 1-0.1 nM, or stronger, e.g., less thanabout 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3, 2, 1, 0.5, 0.2, 0.1,0.05, or 0.01 nM. In an embodiment, the antibody molecule binds to thetarget molecule or cell with a K_(off) slower than 1×10⁻⁴, 5×10⁻⁵, or1×10⁻⁵ s⁻¹. In an embodiment, the antibody molecule binds to the targetmolecule or cell with a K_(on) faster than 1×10⁴, 5×10⁴, 1×10⁵, or 5×10⁵M⁻¹s⁻¹.

In an embodiment, the antibody molecule is capable of inhibiting oractivating a biological function of a target molecule or cell. Forexample, the engineered antibody molecule is capable of inhibiting oractivating a biological function of the target molecule or cell, withthe same, or substantially the same, level of effectiveness, as comparedto the parental antibody molecule, e.g., as determined by IC50, EC50, orLD50.

In an embodiment, the antibody molecule is capable of binding to anepitope on a target molecule or cell. For example, the engineeredantibody molecule is capable of binding to the same, or substantiallythe same, epitope on the target molecule or cell, as compared to theparental antibody molecule.

Exemplary Fusion Proteins

The methods described herein can be used to engineer a variety of fusionproteins, e.g., any Fc fusion protein containing an Fc region.

Exemplary Fc fusion proteins include, but are not limited to, a CTLA-4Fc fusion protein (e.g., belatacept or abatacept), a vascularendothelial growth factor receptor (VEGFR) Fc fusion protein (e.g., aVEGFR1/VEGFR2 Fc fusion protein, e.g., aflibercept or KH902), an IL-1RFc fusion protein (e.g., (rilonacept), a thrombopoietin-binding peptideFc fusion protein (e.g., romiplostim), an LFA-3 Fc fusion protein (e.g.,alefacept), an anti-CD40L Fc fusion protein (e.g., a dimeric fusionprotein comprising the C-terminus of the domain antibody (dAb) targetingthe CD40 ligand (CD40L or CD154) linked to an Fc fragment of IgG1, e.g.,BMS-986004 or letolizumab), an TNF receptor (TNFR) Fc fusion protein(e.g., a recombinant TNF receptor 2 (TNFR2) fused to an IgG1 Fc domain,e.g., OPRX-106 or etanercept), a coagulation Factor VIII-Fc fusionprotein (e.g., BIIB031, efraloctocog-α, or rFVIIIFc), a coagulationFactor IX-Fc Fusion Protein (e.g., BIIB029 or eftrenonacog-α), a FactorIX Fc fusion protein (e.g., rFIXFc), a granulocyte colony-stimulatingfactor Fc fusion protein (e.g., F-627), a follicle stimulating hormone(FSH) Fc fusion protein (e.g., KNO15), an activin type 2B receptor Fcfusion protein (e.g., STM 434), an activin receptor-like kinase 1(ALK-1) inhibitor receptor Fc fusion protein (e.g., dalantercept), anRNase Fc fusion (e.g., RSLV-132), an anti-angiopoietin peptibody (e.g.,a peptide with angiopoietin-binding properties that is fused to the Fcregion, e.g., AMG 386), a tissue nonspecific alkaline phosphatase(TNSALP) Fc fusion protein (e.g., asfotase alfa or ENB-0040), a CD24 Fcfusion protein, a BAFF-Fc fusion protein (e.g., blisibimod), a GLP1peptide analog Fc fusion protein (e.g., dulaglutide or LY2189265), anerythropoietin-mimetic peptide Fc fusion protein (e.g., anerythropoietin-mimetic peptide-IgG1 Fc mimetibody (e.g., CNTO 528), oran erythropoietin-mimetic peptide-IgG4 Fc fusion protein mimetibody(e.g., CNTO 530)), or a CD95 Fc fusion (e.g., APG 101 or apocept).

Animal Models

The polypeptides (e.g., antibody molecules or fusion proteins) describedherein can be evaluated in vivo, e.g., using various animal models. Forexample, an animal model can be used to test the efficacy of apolypeptide (e.g., antibody molecule or fusion proteins) describedherein in modulating a biological function of a target molecule or cell.As another example, an animal model can also be used to test theefficacy of a polypeptide (e.g., antibody molecule) described herein inin treating, preventing, or diagnosing a disorder described hereinAnimal models can also be used, e.g., to investigate for side effects,measure concentrations of antibody molecules in situ, demonstratecorrelations between a function of a target molecule or cell and adisorder described herein.

Exemplary animal models for other disorders described herein are alsoknown in the art. Exemplary types of animals that can be used toevaluate the antibody molecules described herein include, but are notlimited to, mice, rats, rabbits, guinea pigs, and monkeys. Non-humanprimates and transgenic mice expressing human FcRn are typically used asthe model of choice for PK analysis (Avery et al. MAbs. 2016;8(6):1064-78; Fan et al. MAbs. 2016; 8(5):848-53; Tam et al. MAbs. 2013;5(3):397-405).

For example, humanized FcRn mice can be established on the C57BL/6Jbackground in a sequential manner, including the creation of a mousestrain carrying a deletion in the mouse FcRn gene, followed byintroduction of the human FcRn gene. Exemplary mouse lines include,e.g., Tg276 and Tg32 (The Jackson Laboratory stock number 004919 and014565). With further backcrossing and sequential alterations,additional lines can be made. Exemplary mouse models that can be used toevaluate the polypeptides described herein include, but are not limitedto, FcRn-null mice, humanized Tg276 FcRn mice (e.g.,B6.Cg-Fcgrt<tm1Dcr>Tg(CAG-FCGRT)276Dcr/DcrJ with the Jackson Laboratorystock number 004919), humanized Tg32 FcRn mice (e.g.,B6.Cg-Fcgrt<tm1Dcr>Tg(FCGRT)32Dcr/DcrJ with the Jackson Laboratory stocknumber 014565), immunodeficient hFcRn mice (e.g.,B6.Cg-Fcgrt<tm1Dcr>Prkdc<scid>Tg(CAG-FCGRT)276Dcr/DcrJ with the JacksonLaboratory stock number 021146),B6.Cg-Fcgrt<tm1Dcr>Prkdc<scid>Tg(FCGRT)32Dcr/DcrJ with the JacksonLaboratory stock number 018441, andB6.Cg-Rag1<tm>Mom<Fcgrt>tm1Dcr[Tg(CAG-FCGRT)276Dcr/DcrJ with the JacksonLaboratory stock number 16919), e.g., as described in Proetzel et al.BioDrugs. 2014; 28(2): 171-180.

Pharmaceutical Compositions and Kits

In some aspects, this disclosure provides compositions, e.g.,pharmaceutically acceptable compositions, which include a polypeptide(e.g., an antibody molecule or fusion protein) described herein,formulated together with a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, isotonic and absorption delaying agents,and the like that are physiologically compatible. The carrier can besuitable for intravenous, intramuscular, subcutaneous, parenteral,rectal, spinal or epidermal administration (e.g., by injection orinfusion). In certain embodiments, less than about 5%, e.g., less thanabout 4%, 3%, 2%, or 1% of the antibody molecules in the pharmaceuticalcomposition are present as aggregates. In other embodiments, at leastabout 95%, e.g., at least about 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%,or more of the antibody molecules in the pharmaceutical composition arepresent as monomers. In some embodiments, the level of aggregates ormonomers is determined by chromatography, e.g., high performance liquidchromatography size exclusion chromatography (HPLC-SEC).

The compositions set out herein may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes, and suppositories. A suitable form depends onthe intended mode of administration and therapeutic application. Typicalsuitable compositions are in the form of injectable or infusiblesolutions. One suitable mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular). In anembodiment, the polypeptide (e.g., antibody molecule or fusion proteins)is administered by intravenous infusion or injection. In anotherembodiment, the polypeptide (e.g., antibody molecule or fusion proteins)is administered by intramuscular or subcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The polypeptides (e.g., antibody molecules or fusion proteins) describedherein can be administered by a variety of methods. Several are known inthe art, and for many therapeutic, prophylactic, or diagnosticapplications, an appropriate route/mode of administration is intravenousinjection or infusion. For example, the antibody molecules can beadministered by intravenous infusion at a rate of less than 10 mg/min;preferably less than or equal to 5 mg/min to reach a dose of about 1 to100 mg/m², preferably about 5 to 50 mg/m², about 7 to 25 mg/m² and morepreferably, about 10 mg/m². As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. In certain embodiments, the active compoundmay be prepared with a carrier that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, a polypeptide (e.g., an antibody molecule orfusion protein) can be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The antibody molecule (andother ingredients, if desired) may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, theantibody molecule may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. To administer a polypeptide(e.g., an antibody molecule) by other than parenteral administration, itmay be necessary to coat the compound with, or co-administer thecompound with, a material to prevent its inactivation. Therapeutic,prophylactic, or diagnostic compositions can also be administered withmedical devices, and several are known in the art.

Dosage regimens are adjusted to provide the desired response (e.g., atherapeutic, prophylactic, or diagnostic response). For example, asingle bolus may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms aredictated by and directly dependent on (a) the unique characteristics ofthe antibody molecule and the particular therapeutic, prophylactic, ordiagnostic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an antibody molecule for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically,prophylactically, or diagnostically effective amount of an antibodymolecule is about 0.1-50 mg/kg, e.g., about 0.1-30 mg/kg, e.g., about1-30, 1-15, 1-10, 1-5, 5-10, or 1-3 mg/kg, e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, or 50 mg/kg. The antibody molecule can beadministered by intravenous infusion at a rate of less than 10 mg/min,e.g., less than or equal to 5 mg/min to reach a dose of about 1 to 100mg/m², e.g., about 5 to 50 mg/m², about 7 to 25 mg/m², e.g., about 10mg/m². It is to be noted that dosage values may vary with the type andseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed compositions.

The pharmaceutical compositions herein may include a “therapeuticallyeffective amount,” “prophylactically effective amount,” or“diagnostically effectively amount” of a polypeptide (e.g., an antibodymolecule) described herein.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of thepolypeptide (e.g., antibody molecule or fusion protein) may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effect of theantibody molecule is outweighed by the therapeutically beneficialeffects. A “therapeutically effective dosage” typically inhibits ameasurable parameter by at least about 20%, e.g., by at least about 40%,by at least about 60%, or by at least about 80% relative to untreatedsubjects. The measurable parameter may be, e.g., hematuria, coloredurine, foamy urine, pain, swelling (edema) in the hands and feet, orhigh blood pressure. The ability of an antibody molecule to inhibit ameasurable parameter can be evaluated in an animal model systempredictive of efficacy in treating or preventing a disorder describedherein. Alternatively, this property of a composition can be evaluatedby examining the ability of the polypeptide (e.g., antibody molecule orfusion proteins) to modulate a biological function of a target moleculeor cell, e.g., by an in vitro assay.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

A “diagnostically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desireddiagnostic result. Typically, a diagnostically effective amount is onein which a disorder, e.g., a disorder described herein, can be diagnosedin vitro, ex vivo, or in vivo.

Also within this disclosure is a kit that comprises a polypeptide (e.g.,an antibody molecule or fusion protein), described herein. The kit caninclude one or more other elements including: instructions for use;other reagents, e.g., a label, a therapeutic agent, or an agent usefulfor chelating, or otherwise coupling, an antibody molecule to a label ortherapeutic agent, or a radioprotective composition; devices or othermaterials for preparing the polypeptide (e.g., antibody molecule orfusion protein) for administration; pharmaceutically acceptablecarriers; and devices or other materials for administration to asubject.

Nucleic Acids

The present disclosure also features nucleic acids comprising nucleotidesequences that encode polypeptides (e.g., antibody molecules or fusionproteins), e.g., Fc regions of the polypeptides, as described herein.

For example, the present disclosure features a nucleic acid encoding anFc region described herein, e.g., an Fc region comprising one or more ofthe mutations described herein. The Fc region can be engineered from anFc region of an existing polypeptide (e.g., an antibody molecule orfusion protein) described herein. The nucleic acid can comprise anucleotide sequence encoding an amino acid sequence of an Fc region of apolypeptide (e.g., antibody molecule or fusion protein) describedherein, or a nucleotide sequence substantially identical thereto (e.g.,a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or capable of hybridizing under the stringency conditions describedherein).

In an embodiment, the nucleic acid further comprises a nucleotidesequence encoding a heavy chain variable region of a polypeptide (e.g.,an antibody molecule or fusion protein) described herein, or having anucleotide sequence substantially homologous thereto (e.g., a sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, and/orcapable of hybridizing under the stringency conditions describedherein). In another embodiment, the nucleic acid further comprises anucleotide sequence encoding a light chain variable region of apolypeptide (e.g., an antibody molecule or fusion protein) describedherein, or a nucleotide sequence substantially homologous thereto (e.g.,a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or capable of hybridizing under the stringency conditions describedherein). In yet another embodiment, the nucleic acid further comprises anucleotide sequence encoding a heavy chain variable region and a lightchain variable region of a polypeptide (e.g., an antibody molecule orfusion protein) described herein, or a nucleotide sequence substantiallyhomologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or capable of hybridizing under thestringency conditions described herein).

In an embodiment, the nucleic acid further comprises a nucleotidesequence encoding at least one, two, or three CDRs from a heavy chainvariable region of a polypeptide (e.g., an antibody molecule or fusionprotein) described herein, or a nucleotide sequence substantiallyhomologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or capable of hybridizing under thestringency conditions described herein). In another embodiment, thenucleic acid further comprises a nucleotide sequence encoding at leastone, two, or three CDRs from a light chain variable region of apolypeptide (e.g., an antibody molecule or fusion protein) describedherein, or a nucleotide sequence substantially homologous thereto (e.g.,a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or capable of hybridizing under the stringency conditions describedherein). In yet another embodiment, the nucleic acid comprises anucleotide sequence encoding at least one, two, three, four, five, orsix CDRs from heavy and light chain variable regions of a polypeptide(e.g., an antibody molecule or fusion protein) described herein, or anucleotide sequence substantially homologous thereto (e.g., a sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, and/orcapable of hybridizing under the stringency conditions describedherein).

In an embodiment, the nucleic acid comprises a portion of a nucleotidesequence described herein. The portion may encode, for example, an Fcregion, a variable region (e.g., VH or VL); one, two, or three or more(e.g., four, five, or six) CDRs; or one, two, three, or four or moreframework regions.

The nucleic acids disclosed herein include deoxyribonucleotides orribonucleotides, or analogs thereof. The polynucleotide may be eithersingle-stranded or double-stranded, and if single-stranded may be thecoding strand or non-coding (antisense) strand. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The nucleic acid may be a recombinant polynucleotide, or apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich either does not occur in nature or is linked to anotherpolynucleotide in a non-natural arrangement.

In some aspects, the application features host cells and vectorscontaining the nucleic acids described herein. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell, as described in more detail below.

Vectors

The present disclosure features vectors that comprise nucleotidesequences encoding polypeptides (e.g., an antibody molecules or fusionproteins), e.g., Fc regions of the polypeptides, as described herein.

For example, the present disclosure features a vector comprising anucleotide sequence encoding an Fc region described herein, e.g., an Fcregion comprising one or more of the mutations described herein. The Fcregion can be engineered from an Fc region of an existing polypeptide(e.g., an antibody molecule or fusion protein) described herein. Thevector can comprise a nucleotide sequence encoding an amino acidsequence of an Fc region of a polypeptide (e.g., antibody molecule orfusion protein) described herein, or a nucleotide sequence substantiallyidentical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% ormore identical thereto, and/or capable of hybridizing under thestringency conditions described herein).

The vectors include, but are not limited to, a virus, plasmid, cosmid,lambda phage or a yeast artificial chromosome (YAC).

Numerous vector systems can be employed. For example, one class ofvectors utilizes DNA elements which are derived from animal viruses suchas, for example, bovine papilloma virus, polyoma virus, adenovirus,vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV orMOMLV) or SV40 virus. Another class of vectors utilizes RNA elementsderived from RNA viruses such as Semliki Forest virus, Eastern EquineEncephalitis virus and Flaviviruses.

Additionally, cells which have stably integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow for the selection of transfected host cells. The marker mayprovide, for example, prototropy to an auxotrophic host, biocideresistance (e.g., antibiotics), or resistance to heavy metals such ascopper, or the like. The selectable marker gene can be either directlylinked to the DNA sequences to be expressed, or introduced into the samecell by cotransformation. Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include splice signals, aswell as transcriptional promoters, enhancers, and termination signals.

Once the expression vector or DNA sequence containing the constructs hasbeen prepared for expression, the expression vectors may be transfectedor introduced into an appropriate host cell. Various techniques may beemployed to achieve this, such as, for example, protoplast fusion,calcium phosphate precipitation, electroporation, retroviraltransduction, viral transfection, gene gun, lipid based transfection orother conventional techniques. In the case of protoplast fusion, thecells are grown in media and screened for the appropriate activity.

Methods and conditions for culturing the resulting transfected cells andfor recovering the polypeptide (e.g., antibody molecule) produced areknown to those skilled in the art, and may be varied or optimizeddepending upon the specific expression vector and mammalian host cellemployed, based upon the present description.

Cells

The present disclosure also provides host cells comprising a nucleicacid encoding a polypeptide (e.g., an antibody molecule or fusionprotein) as described herein. The polypeptide (e.g., antibody moleculeor fusion protein) can be engineered in accordance with a methoddescribed herein. For example, the host cells may comprise a nucleicacid molecule having a nucleotide sequence of a polypeptide describedherein (e.g., an antibody molecule or fusion protein described herein),a sequence substantially homologous thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or capable ofhybridizing under the stringency conditions described herein), or aportion of one of said nucleic acids.

In some embodiments, the host cells are genetically engineered tocomprise nucleic acids encoding the polypeptide (e.g., antibody moleculeor fusion protein) described herein.

In certain embodiments, the host cells are genetically engineered byusing an expression cassette. The phrase “expression cassette,” refersto nucleotide sequences, which are capable of affecting expression of agene in hosts compatible with such sequences. Such cassettes may includea promoter, an open reading frame with or without introns, and atermination signal. Additional factors necessary or helpful in effectingexpression may also be used, such as, for example, an induciblepromoter.

The disclosure also provides host cells comprising the vectors describedherein.

The cell can be, but is not limited to, a eukaryotic cell, a bacterialcell, an insect cell, or a human cell. Suitable eukaryotic cellsinclude, but are not limited to, Vero cells, HeLa cells, COS cells, CHOcells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cellsinclude, but are not limited to, Sf9 cells.

Uses of Polypeptides

The polypeptides (e.g., antibody molecules or fusion proteins) disclosedherein, as well as the pharmaceutical compositions disclosed herein,have in vitro, ex vivo, and in vivo therapeutic, prophylactic, and/ordiagnostic utilities.

In an embodiment, the polypeptide (e.g., antibody molecule or fusionprotein) modulates (e.g., reduces (e.g., inhibits, blocks, orneutralizes) or increases (e.g., activates, initiates, or enhances)) oneor more biological activities of a target molecule (e.g., protein) orcell. For example, these polypeptides (e.g., antibody molecules orfusion proteins) can be administered to cells in culture, in vitro or exvivo, or to a subject, e.g., a human subject, e.g., in vivo, to modulateone or more biological activities of the target molecule or cell.Accordingly, in an aspect, the disclosure provides a method of treating,preventing, or diagnosing a disorder, e.g., a disorder described herein,in a subject, comprising administering to the subject a polypeptide(e.g., an antibody molecule or fusion protein) described herein, suchthat the disorder is treated, prevented, or diagnosed. For example, thedisclosure provides a method comprising contacting the polypeptide(e.g., antibody molecule or fusion protein) described herein with cellsin culture, e.g. in vitro or ex vivo, or administering the polypeptide(e.g., antibody molecule or fusion protein) described herein to asubject, e.g., in vivo, to treat, prevent, or diagnose a disorder, e.g.,a disorder associated with a target molecule or cell (e.g., a disorderdescribed herein).

As used herein, the term “subject” is intended to include human andnon-human animals. In some embodiments, the subject is a human subject,e.g., a human patient having a disorder described herein, or at risk ofhaving a disorder described herein. The term “non-human animals”includes mammals and non-mammals, such as non-human primates. In anembodiment, the subject is a human. The methods and compositionsdescribed herein are suitable for treating human patients for a disorderdescribed herein. Patients having a disorder described herein includethose who have developed a disorder described herein but are (at leasttemporarily) asymptomatic, patients who have exhibited a symptom of adisorder described herein, or patients having a disorder related to orassociated with a disorder described herein.

Methods of Treating or Preventing Disorders

The polypeptides (e.g., antibody molecules or fusion proteins) describedherein can be used to treat or prevent disorders or conditions. In anembodiment, the polypeptide has an optimal or improved half-life, whichcan be desirable for treating or preventing the disorder or condition.While not wishing to be bound by theory, it is believed that in anembodiment, the polypeptide described herein (e.g., the polypeptidehaving an optimal or improved half-life) can provide one or morebenefits over another polypeptide having the same or similar bindingaffinity and/or specificity (e.g., a polypeptide that does not have, orhas not been engineered to have, an optimal or improved half-life).These benefits can include, but are not limited to, an increasedtherapeutic or preventive efficacy, a reduced dosage regimen, or animproved pharmacokinetic property. In an embodiment, the polypeptideincludes a mutated Fc region as described herein.

Exemplary disorders or conditions that can be treated or prevented bythe polypeptides described herein include, but are not limited to, acancer (e.g., a solid tumor or a hematologic cancer), an infectiousdisease (e.g., a bacterial infection or a viral infection), an immunedisorder (e.g., an autoimmune disorder), an inflammatory disorder, ametabolic disorder (e.g., diabetes), a cardiovascular disorder, an organtransplant rejection. In an embodiment, the disorder is a chronicdisorder.

Exemplary cancers that can be treated or prevented by the polypeptidesdescribed herein include, but are not limited to, acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma,Kaposi sarcoma, an AIDS-related lymphoma, primary central nervous system(CNS) lymphoma, anal cancer, appendix cancer, astrocytoma, atypicalteratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladdercancer, bone cancer (e.g., Ewing sarcoma or osteosarcoma and malignantfibrous histiocytoma), brain tumor (e.g., astrocytomas, brain stemglioma, central nervous system atypical teratoid/rhabdoid tumor, centralnervous system embryonal tumor, central nervous system germ cell tumor,craniopharyngioma, or ependymoma), breast cancer, bronchial tumor,Burkitt lymphoma, carcinoid tumor (e.g., gastrointestinal carcinoidtumor), cardiac (heart) tumor, embryonal tumor, germ cell tumor,lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronicmyeloproliferative neoplasm, colon cancer, colorectal cancer,craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ(DCIS), endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor,extragonadal germ cell tumor, eye cancer (e.g., intraocular melanoma orretinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone,osteosarcoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumors(GIST), germ cell tumor (e.g., central nervous system tumor,extracranial tumor, extragonadal tumor, ovarian cancer, or testicularcancer), gestational trophoblastic disease, glioma, hairy cell leukemia,head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma,hypopharyngeal cancer, intraocular melanoma, islet cell tumor,pancreatic neuroendocrine tumor, Kaposi sarcoma, kidney cancer (e.g.,renal cell cancer or Wilms tumor), Langerhans cell histiocytosis (LCH),laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia (ALL),acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), or hairy cell leukemia), lip andoral cavity cancer, liver cancer, lung cancer (e.g., non-small cell lungcancer (NSCLC) or small cell lung cancer), lymphoma (e.g., aids-related,Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma,non-Hodgkin lymphoma, or primary central nervous system (CNS) lymphoma),Waldenström macroglobulinemia, male breast cancer, malignant fibroushistiocytoma of bone and osteosarcoma, melanoma (e.g., intraocular (eye)melanoma), Merkel cell carcinoma, mesothelioma, metastatic squamous neckcancer, midline tract carcinoma, mouth cancer, multiple endocrineneoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosisfungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferativeneoplasm, chronic myeloproliferative neoplasm, nasal cavity andparanasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oralcancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcomaand malignant fibrous histiocytoma of bone, ovarian cancer (e.g.,epithelial ovarian cancer or germ cell ovarian tumor), pancreaticcancer, pancreatic neuroendocrine tumors (islet cell tumors),papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pituitary tumor, pleuropulmonary blastoma, peritoneal cancer, prostatecancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary glandcancer, sarcoma (e.g., Ewing sarcoma, Kaposi sarcoma, osteosarcoma,rhabdomyosarcoma, soft tissue sarcoma, or uterine sarcoma), Sezarysyndrome, skin cancer (e.g., melanoma, Merkel cell carcinoma, ornonmelanoma skin cancer), small intestine cancer, squamous cellcarcinoma, testicular cancer, throat cancer, thymoma and thymiccarcinoma, thyroid cancer, transitional cell cancer of the renal pelvisand ureter, urethral cancer, endometrial uterine cancer, vaginal cancer,vulvar cancer, or a metastatic lesion thereof.

Exemplary infectious diseases that can be treated or prevented by thepolypeptides described herein include, but are not limited to,Acinetobacter infections, actinomycosis, African sleeping sickness(African trypanosomiasis), AIDS (acquired immunodeficiency syndrome),amebiasis, anaplasmosis, angiostrongyliasis, anisakiasis, anthrax,arcanobacterium haemolyticum infection, argentine hemorrhagic fever,ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacilluscereus infection, bacterial pneumonia, bacterial vaginosis, bacteroidesinfection, balantidiasis, bartonellosis, baylisascaris infection, bkvirus infection, black piedra, blastocystosis, blastomycosis, bolivianhemorrhagic fever, botulism (and infant botulism), brazilian hemorrhagicfever, brucellosis, bubonic plague, burkholderia infection, buruliulcer, calicivirus infection (norovirus and sapovirus),campylobacteriosis, candidiasis (moniliasis; thrush), capillariasis,carrion's disease, cat-scratch disease, cellulitis, chagas disease(american trypanosomiasis), chancroid, chickenpox, chikungunya,chlamydia, chlamydophila pneumoniae infection (taiwan acute respiratoryagent or twar), cholera, chromoblastomycosis, chytridiomycosis,clonorchiasis, Clostridium difficile colitis, coccidioidomycosis,colorado tick fever (CTF), common cold (Acute viral rhinopharyngitis;Acute coryza), Creutzfeldt-Jakob disease (CJD), Crimean-Congohemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneouslarva migrans (CLM), cyclosporiasis, cysticercosis, cytomegalovirusinfection, dengue fever, desmodesmus infection, dientamoebiasis,diphtheria, diphyllobothriasis, dracunculiasis, ebola hemorrhagic fever,echinococcosis, ehrlichiosis, enterobiasis (pinworm infection),enterococcus infection, enterovirus infection, epidemic typhus, erythemainfectiosum (fifth disease), exanthem subitum (sixth disease),fasciolasis, fasciolopsiasis, fatal familial insomnia (FFI), filariasis,food poisoning by Clostridium perfringens, free-living amebic infection,fusobacterium infection, gas gangrene (clostridial myonecrosis),geotrichosis, gerstmann-straussler-scheinker syndrome (GSS), giardiasis,glanders, gnathostomiasis, gonorrhea, granuloma inguinale (donovanosis),Group A streptococcal infection, Group B streptococcal infection,Haemophilus influenzae infection, hand, foot and mouth disease (HFMD),Hantavirus Pulmonary Syndrome (HPS), heartland virus disease,Helicobacter pylori infection, hemolytic-uremic syndrome (HUS),hemorrhagic fever with renal syndrome (HFRS), hepatitis A, hepatitis B,hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis,hookworm infection, human bocavirus infection, human ewingiiehrlichiosis, human granulocytic anaplasmosis (HGA), humanmetapneumovirus infection, Human monocytic ehrlichiosis, humanpapillomavirus (HPV) infection, Human parainfluenza virus infection,Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono),influenza (flu), isosporiasis, kawasaki disease, keratitis, kingellakingae infection, kuru, lassa fever, legionellosis (legionnaires'disease), legionellosis (pontiac fever), leishmaniasis, leprosy,leptospirosis, listeriosis, lyme disease (lyme borreliosis), lymphaticfilariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria,Marburg hemorrhagic fever (MHF), Measles, Middle East respiratorysyndrome (MERS), melioidosis (Whitmore's disease), meningitis,meningococcal disease, metagonimiasis, microsporidiosis, molluscumcontagiosum (MC), Monkeypox, Mumps, Murine typhus (Endemic typhus),Mycoplasma pneumonia, Mycetoma (disambiguation), Myiasis, Neonatalconjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakobdisease (vCJD, nvCJD), nocardiosis, onchocerciasis (River blindness),opisthorchiasis, paracoccidioidomycosis (South American blastomycosis),paragonimiasis, pasteurellosis, pediculosis capitis (head lice),pediculosis corporis (body lice), pediculosis pubis (pubic lice, crablice), pelvic inflammatory disease (PID), pertussis (Whooping cough),plague, pneumococcal infection, pneumocystis pneumonia (PCP), pneumonia,poliomyelitis, prevotella infection, primary amoebic meningoencephalitis(PAM), progressive multifocal leukoencephalopathy, psittacosis, Q fever,rabies, relapsing fever, respiratory syncytial virus infection,rhinosporidiosis, rhinovirus infection, rickettsial infection,rickettsialpox, Rift Valley fever (RVF), Rocky Mountain spotted fever(RMSF), rotavirus infection, rubella, salmonellosis, SARS (Severe AcuteRespiratory Syndrome), scabies, schistosomiasis, sepsis, shigellosis(Bacillary dysentery), shingles (Herpes zoster), smallpox (Variola),sporotrichosis, staphylococcal food poisoning, staphylococcal infection,strongyloidiasis, subacute sclerosing panencephalitis, syphilis,Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber's itch), Tineacapitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the Body),Tinea cruris (Jock itch), Tinea manum (Ringworm of the Hand), Tineanigra, Tinea pedis (Athlete's foot), Tinea unguium (Onychomycosis),Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular LarvaMigrans (OLM)), Toxocariasis (Visceral Larva Migrans (VLM)), Trachoma,Toxoplasmosis, Trichinosis, Trichomoniasis, Trichuriasis (Whipworminfection), Tuberculosis, Tularemia, Typhoid fever, Typhus fever,Ureaplasma urealyticum infection, Valley fever, Venezuelan equineencephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus infection,Vibrio parahaemolyticus enteritis, viral pneumonia, West Nile Fever,white piedra (Tinea blanca), Yersinia pseudotuberculosis infection,yersiniosis, yellow fever, Zika fever, or zygomycosis.

Exemplary immune disorders or conditions that can be treated orprevented by the polypeptides described herein include, but are notlimited to, Addison's disease, agammaglobulinemia, alopecia areata,amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis,antiphospholipid syndrome (APS), autoimmune hepatitis, autoimmune innerear disease (AIED), axonal & neuronal neuropathy (AMAN), Behcet'sdisease, Bullous pemphigoid, Castleman disease (CD), Celiac disease,Chagas disease, chronic inflammatory demyelinating polyneuropathy(CIDP), chronic recurrent multifocal osteomyelitis (CRMO),Churg-Strauss, Cicatricial pemphigoid/benign mucosal pemphigoid, Cogan'ssyndrome, Cold agglutinin disease, Congenital heart block, Coxsackiemyocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis,dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus,Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE),eosinophilic fasciitis, erythema nodosum, essential mixedcryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis,giant cell arteritis (temporal arteritis), giant cell myocarditis,Glomerulonephritis, Goodpasture's syndrome, Granulomatosis withPolyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto'sthyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), herpesgestationis or pemphigoid gestationis (PG), hypogammalglobulinemia, IgAnephropathy, IgG4-related sclerosing disease, inclusion body myositis(IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes(Type 1 diabetes), juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, linear IgA disease (LAD),lupus, Lyme disease chronic, Meniere's disease, microscopic polyangiitis(MPA), mixed connective tissue disease (MCTD), Mooren's ulcer,Mucha-Habermann disease, multiple sclerosis (MS), Myasthenia gravis,Myositis, Narcolepsy, Neuromyelitis optica, neutropenia, ocularcicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR),PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paraneoplastic cerebellar degeneration (PCD), Paroxysmalnocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis(peripheral uveitis), Parsonnage-Turner syndrome, Pemphigus, peripheralneuropathy, Perivenous encephalomyelitis, pernicious anemia (PA), POEMSsyndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonalgammopathy, skin changes), polyarteritis nodosa, polymyalgia rheumatica,polymyositis, postmyocardial infarction syndrome, postpericardiotomysyndrome, primary biliary cirrhosis, primary sclerosing cholangitis,progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cellaplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, ReactiveArthritis, Reflex sympathetic dystrophy, Reiter's syndrome, relapsingpolychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis,rheumatic fever, rheumatoid arthritis (RA), sarcoidosis, Schmidtsyndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicularautoimmunity, Stiff person syndrome (SPS), subacute bacterialendocarditis (SBE), Susac's syndrome, sympathetic ophthalmia (SO),Takayasu's arteritis, temporal arteritis/Giant cell arteritis,thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transversemyelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiatedconnective tissue disease (UCTD), uveitis, vasculitis, vitiligo, orWegener's granulomatosis (Granulomatosis with Polyangiitis (GPA)).

The polypeptides (e.g., antibody molecules or fusion proteins) describedherein are typically administered at a frequency that keeps atherapeutically effective level of polypeptides in the patient's systemuntil the patient recovers. For example, the polypeptides may beadministered at a frequency that achieves a serum concentrationsufficient for at least about 1, 2, 5, 10, 20, 30, or 40 polypeptides tobind each target molecule or cell. In an embodiment, the polypeptidesare administered every 1, 2, 3, 4, 5, 6, or 7 days, every 1, 2, 3, 4, 5,or 6 weeks, or every 1, 2, 3, 4, 5, or 6 months.

Methods of administering various polypeptides (e.g., antibody moleculesor fusion proteins) are known in the art and are described below.Suitable dosages of the polypeptides used will depend on the age andweight of the subject and the particular drug used.

The polypeptides can be used by themselves or conjugated to a secondagent, e.g., an bacterial agent, toxin, or protein, e.g., a secondpolypeptide. This method includes: administering the polypeptide, aloneor conjugated to a second agent, to a subject requiring such treatment.The polypeptides can be used to deliver a variety of therapeutic agents,e.g., a toxin, or mixtures thereof.

Combination Therapies

The polypeptides (e.g., antibody molecules or fusion proteins) can beused in combination with other therapies. For example, the combinationtherapy can include a polypeptide co-formulated with, and/orco-administered with, one or more additional therapeutic agents, e.g.,one or more additional therapeutic agents described herein. In otherembodiments, the polypeptides are administered in combination with othertherapeutic treatment modalities, e.g., other therapeutic treatmentmodalities described herein. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

Administered “in combination”, as used herein, means that two (or more)different treatments are delivered to the subject before, or during thecourse of the subject's affliction with a disorder. In an embodiment,two or more treatments are delivered prophylactically, e.g., before thesubject has the disorder or is diagnosed with the disorder. In anotherembodiment, the two or more treatments are delivered after the subjecthas developed or diagnosed with the disorder. In some embodiments, thedelivery of one treatment is still occurring when the delivery of thesecond begins, so that there is overlap. This is sometimes referred toherein as “simultaneous” or “concurrent delivery.” In other embodiments,the delivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

In an embodiment, the polypeptide is administered in combination with asecond therapy (e.g., an additional agent) to treat or prevent adisorder described herein. In an embodiment, the additional agent is asecond polypeptide (e.g., antibody molecule), e.g., a polypeptide (e.g.,an antibody molecule) different from a first polypeptide (e.g., antibodymolecule). Exemplary polypeptides (e.g., antibody molecules) that can beused in combination include, but are not limited to, any combination ofthe polypeptides (e.g., antibody molecules) described herein. In anotherembodiment, the additional agent is other than a polypeptide (e.g.,antibody molecule). For example, the additional agent can be a smallmolecule or a nucleic acid molecule. In yet another embodiment, thesecond therapy is chosen from a surgery, a radiation therapy, a celltherapy (e.g., a stem cell therapy), or an organ or tissuetransplantation.

In an embodiment, the second therapy comprises a therapy chosen from oneor more of: an androgen replacement therapy, an antihormone therapy, anantiserum therapy, an autologous immune enhancement therapy, abiotherapy, a blood irradiation therapy, a brachytherapy, a cardiacresynchronization therapy, a cell therapy, a cell transfer therapy, achelation therapy, a chemotherapy, a chrysotherapy, a cobalt therapy, acold compression therapy, a cryotherapy, an electroconvulsive therapy,an electromagnetic therapy, an electron therapy, an electrotherapy, anenzyme replacement therapy, an epigenetic therapy, an estrogenreplacement therapy, an extracorporeal shockwave therapy, a fast neutrontherapy, a fluoride therapy, a gene therapy, a heat therapy, ahelminthic therapy, a hormone therapy, a hormone replacement therapy, ahost modulatory therapy, a hyperbaric oxygen therapy, a hyperthermiatherapy, an immunosuppressive therapy, an immunotherapy, anintraoperative electron radiation therapy, an intraoperative radiationtherapy, an inversion therapy, a laser therapy, a light therapy, alithium therapy, a low level laser therapy, a magnet therapy, a magneticresonance therapy, a medical gas therapy, a medical nutrition therapy, amolecular chaperone therapy, a molecular therapy, a monoclonal antibodytherapy, a negative air ionization therapy, a neutron capture therapy, aneutron therapy, an oral rehydration therapy, an osmotherapy, an oxygentherapy, an ozone therapy, a palliative therapy, a particle therapy, aphage therapy, a phonemic neurological hypochromium therapy, aphotodynamic therapy, a phototherapy, a photothermal therapy, a physicaltherapy, a prototherapy, a protein therapy, a proton therapy, a pulsedelectromagnetic field therapy, a PUVA therapy, a radiation therapy, arehydration therapy, a respiratory therapy, salvage therapy, aserotherapy, a stem cell therapy, a stereotactic radiation therapy, atargeted therapy, a thermotherapy, a TK cell therapy, a tolerogenictherapy, a transdermal continuous oxygen therapy, an ultraviolet lighttherapy, or a virotherapy.

Exemplary therapies that can be used in combination with a polypeptideor composition described herein to treat or prevent other disorders arealso described in the section of “Methods of Treating or PreventingDisorders” herein.

Methods of Diagnosis

In some aspects, the present disclosure provides a diagnostic method fordetecting the presence of a target molecule (e.g., a protein) or cell invitro (e.g., in a biological sample, such as a biopsy or body fluid(e.g., blood, urine, or cerebrospinal fluid) sample) or in vivo (e.g.,in vivo imaging in a subject). The method includes: (i) contacting thesample with a polypeptide described herein (e.g., an antibody moleculedescribed herein), or administering to the subject, the polypeptide(e.g., antibody molecule); (optionally) (ii) contacting a referencesample, e.g., a control sample (e.g., a control biological sample, suchas a biopsy or body fluid (e.g., blood, urine, or cerebrospinal fluid)sample) or a control subject with a polypeptide described herein (e.g.,an antibody molecule described herein); and (iii) detecting formation ofa complex between the polypeptide (e.g., antibody molecule) and thetarget molecule or cell in the sample or subject, or the control sampleor subject, wherein a change, e.g., a statistically significant change,in the formation of the complex in the sample or subject relative to thecontrol sample or subject is indicative of the presence of the targetmolecule or cell in the sample. The polypeptide (e.g., antibodymolecule) can be directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound polypeptide(e.g., antibody molecule). Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials, as described herein.

The term “sample,” as it refers to samples used for detecting bacteriaincludes, but is not limited to, cells, cell lysates, proteins ormembrane extracts of cells, body fluids such as blood, urine, or CSF, ortissue samples such as biopsies.

Complex formation between the polypeptide (e.g., antibody molecule), andthe target molecule or cell, can be detected by measuring or visualizingeither the polypeptide (e.g., antibody molecule) bound to the targetmolecule or cell, or unbound polypeptide (e.g., antibody molecule). Anysuitable detection assays can be used, and conventional detection assaysinclude an enzyme-linked immunosorbent assays (ELISA), aradioimmunoassay (RIA) or tissue immunohistochemistry. Alternative tolabeling the polypeptide, the presence of the target molecule or cellcan be assayed in a sample by a competition immunoassay utilizingstandards labeled with a detectable substance and an unlabeledpolypeptide. In this assay, the biological sample, the labeled standardsand the polypeptide are combined and the amount of labeled standardbound to the unlabeled binding molecule is determined. The amount of thetarget molecule or cell in the sample is inversely proportional to theamount of labeled standard bound to the polypeptide (e.g., antibodymolecule).

The polypeptides (e.g., antibody molecules) described herein can be usedto diagnose disorders that can be treated or prevented by thepolypeptides described herein. The detection or diagnostic methodsdescribed herein can be used in combination with other methods describedherein to treat or prevent disorders described herein.

The present disclosure also includes any of the following numberedparagraphs:

1. A polypeptide comprising an Fc region, wherein the Fc regioncomprises a mutation, wherein the polypeptide has 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or all of the following properties:

a) has an increased binding affinity for a neonatal Fc receptor (FcRn)at a pH between 6.0 and 6.5, compared to a reference polypeptide;

b) has a higher binding affinity for FcRn at a pH between 6.0 and 6.5than the binding affinity at a pH between 7.0 and 7.4;

c) binds to an FcRn at a pH between 6.0 and 6.5 with a dissociationconstant (K_(d)) of 300 nM or less;

d) binds to the FcRn at a pH between 7.0 and 7.4 with a K_(d) of 50 nMor more;

e) has the same, substantially the same, or increased binding affinityfor an Fcγ receptor, compared to a reference polypeptide;

f) has the same, or substantially the same, thermal stability, comparedto a reference polypeptide;

g) has the same, substantially the same, or increased binding affinityfor C1q, compared to a reference polypeptide;

h) has the same, substantially the same, or increased binding affinityfor TRIM21, compared to a reference polypeptide.

i) has an effector function that is the same, substantially the same, orincreased, compared to a reference polypeptide;

j) has an increased half-life in vivo, compared to a referencepolypeptide;

k) has a biological function, in vitro, ex vivo, or in vivo, that is thesame, substantially the same, or increased, compared to a referencepolypeptide;

l) has a developability characteristic that is the same or substantiallythe same, compared to a reference polypeptide;

m) has the same, substantially the same, or increased binding affinity,specificity, or both, for an epitope, compared to a referencepolypeptide; or

n) increases mucosal uptake, compared to a reference polypeptide, and

wherein the polypeptide has at least properties a), b), and one, two,three, four, or all of properties e), f), g), h), or i).

2. The polypeptide of paragraph 1, which has at least properties a), b),c), d), and one, two, three, four, or all of properties e), f), g), h),or i).

3. The polypeptide of paragraph 1 or 2, which has at least propertiesa), b), one, two, three, four, or all of properties e), f), g), h), ori), and one, two, three, four, five, six, or all of properties c), d),j), k), l), m), or n).

4. The polypeptide of any of paragraphs 1-3, which has at least 1.5, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50-fold increase in thebinding affinity for the FcRn at pH 6.0, compared to a referencepolypeptide, as determined by an octet-based assay or a cell-basedassay.

5. The polypeptide of any of paragraphs 1-4, which has at least 1.5, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 50-fold higher in the bindingaffinity for the FcRn at pH 6.0 than that at pH 7.4, as determined by anoctet-based assay or a cell-based assay.

6. The polypeptide of any of paragraphs 1-5, which binds to the FcRn atpH 6.0 with a dissociation constant (K_(d)) of 250 nM or less, 200 nM orless, 150 nM or less, 100 nM or less, 50 nM or less, 25 nM or less, 10nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less,0.2 nM or less, 0.1 nM or less, 0.05 nM or less, 0.02 nM or less, 0.01nM or less, between 25 nM and 0.1 nM, between 20 nM and 0.5 nM, between15 nM and 1 nM, between 10 nM and 5 nM, or between 20 nM and 10 nM, asdetermined by an octet-based assay or a cell-based assay.

7. The polypeptide of any of paragraphs 1-6, which binds to the FcRn atpH 7.4 with a K_(d) of 60 nM or more, 80 nM or more, 100 nM or more, 150nM or more, 200 nM or more, 500 nM or more, between 50 nM and 500 nM, orbetween 100 nM and 250 nM, as determined by an octet-based assay or acell-based assay.

8. The polypeptide of any of paragraphs 1-7, which decreases the bindingaffinity for one, two, or, all of FcγRI, FcγRIIa/b, or FcγRIII by nomore than 10%, 20%, 30%, 40%, or 50%, or increases the binding affinityfor one, two, or all of FcγRI, FcγRIIa/b, or FcγRIII by at least 1.5, 2,3, 4, or 5-fold, compared to a reference polypeptide, as determined byan octet-based assay or a cell-based assay.

9. The polypeptide of any of paragraphs 1-8, which increases ordecreases the melting temperature by no more than 1° C., 2° C., 3° C.,4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C., compared to areference polypeptide, as determined by a sypro orange assay.

10. The polypeptide of any of paragraphs 1-9, which decreases thebinding affinity for C1q by no more than 10%, 20%, 30%, 40%, or 50%, orincreases the binding affinity for C1q by at least 1.5, 2, 3, 4, or5-fold, compared to a reference polypeptide, as determined by ELISA.

11. The polypeptide of any of paragraphs 1-10, which decreases thebinding affinity for TRIM21 by no more than 10%, 20%, 30%, 40%, or 50%,or increases the binding affinity for TRIM21 by at least 1.5, 2, 3, 4,or 5-fold, compared to a reference polypeptide, as determined by ELISA.

12. The polypeptide of any of paragraphs 1-11, which decreases one, two,three, or all of a complement dependent cytotoxicity (CDC), an antibodydependent cell mediated cytotoxicity (ADCC), an antibody dependent cellmediated phagocytosis (ADCP), or an antibody dependent intracellularneutralization (ADIN) by no more than 10%, 20%, 30%, 40%, or 50%, orincreases one, two, three, or all of a complement dependent cytotoxicity(CDC), an antibody dependent cell mediated cytotoxicity (ADCC), anantibody dependent cell mediated phagocytosis (ADCP), or an antibodydependent intracellular neutralization (ADIN) by at least 1.5, 2, 3, 4,or 5-fold, compared to a reference polypeptide.

13. The polypeptide of any of paragraphs 1-12, which has at least 1.5,2, 3, 4, 5, 6, 7, 8, 9, or 10-fold increase in the half-life in vivo,compared to a reference polypeptide, as determined in an animal model.

14. The polypeptide of any of paragraphs 1-13, which decreases thebiological function, in vitro, ex vivo, or in vivo by no more than 10%,20%, 30%, 40%, or 50%, or increases the biological function, in vitro,ex vivo, or in vivo by at least 1.5, 2, 3, 4, or 5-fold, compared to areference polypeptide.

15. The polypeptide of any of paragraphs 1-14, which alters one, two,three, or all of stability, solubility, aggregation, or expression levelby no more than 10%, 20%, 30%, 40%, or 50%, compared to a referencepolypeptide.

16. The polypeptide of any of paragraphs 1-15, which decreases thebinding affinity, specificity, or both by no more than 10%, 20%, 30%,40%, or 50%, or increases the binding affinity, specificity, or both byat least 1.5, 2, 3, 4, or 5-fold, compared to a reference polypeptide.

17. The polypeptide of any of paragraphs 1-16, which increases mucosaluptake by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, compared toa reference polypeptide, as determined by a transcytosis assay.

18. The polypeptide of any of paragraphs 1-4 or 8-17, wherein thereference polypeptide comprises a wild-type Fc region, an Fc regioncomprising the amino acid sequence of SEQ ID NO: 1, or an amino acidsequence at least about 85%, 90%, 95%, 99% or more identical thereto, orwhich differs by no more than 1, 2, 5, 10, or 15 amino acid residues.

19. The polypeptide of any of paragraphs 1-18, wherein the mutation isin a residue in a CH2 domain.

20. The polypeptide of any of paragraphs 1-18, wherein the mutation isin a residue in a CH3 domain.

21. The polypeptide of any of paragraphs 1-20, comprising at least onemutation in a residue in a CH2 domain and at least one mutation in aresidue in a CH3 domain.

22. The polypeptide of any of paragraphs 1-21, further comprising amutation in a residue in a region other than a CH2 domain and/or a CH3domain.

23. The polypeptide of any of paragraphs 1-22, wherein the mutation doesnot alter, or does not substantially alter, the conformation of thelinker region between a CH2 domain and a CH3 domain.

24. The polypeptide of any of paragraphs 1-23, wherein the mutation doesnot introduce 3, 4, 5, 6, 7, 8, 9, 10, or more consecutive hydrophobicor aromatic residues on a surface region.

25. The polypeptide of any of paragraphs 1-24, which comprises anantibody molecule.

26. The polypeptide of any of paragraphs 1-25, which comprises an IgG.

27. The polypeptide of any of paragraphs 1-26, which comprises an IgG1,IgG2, IgG3, or IgG4.

28. The polypeptide of any of paragraphs 1-27, which comprises a heavychain immunoglobulin variable region, a light chain immunoglobulinvariable region, or both.

29. The polypeptide of any of paragraphs 1-28, which comprises atetramer of two heavy chain immunoglobulin variable regions and twolight chain immunoglobulin variable regions.

30. The polypeptide of any of paragraphs 1-29, which comprises a fulllength antibody molecule.

31. The polypeptide of any of paragraphs 1-30, which comprises afragment of an antibody molecule.

32. The polypeptide of any of paragraphs 1-31, which comprises achimeric antibody molecule or a murine antibody molecule.

33. The polypeptide of any of paragraphs 1-32, which comprises a humanantibody molecule or a humanized antibody molecule.

34. The polypeptide of any of paragraphs 1-24, which comprises a fusionprotein.

35. The polypeptide of any of paragraphs 1-34, comprising 1, 2, 3, 4, orall of the following:

(i) a mutation in a residue in a surface region that interacts withFcRn;

(ii) a mutation in a residue that is a peripheral residue along theFc-FcRn interface;

(iii) a mutation is in a residue that is non-contact residue in Fc-FcRnbinding;

(iv) a mutation in a residue which is a helix contact reside thatenhances the conformational dynamics of a helix comprising 1, 2, 3, 4,5, or all of P247, K248, D249, T250, L251, or M252; or

(v) a mutation, which modulates pK of a histidine or is an introductionof a histidine along the Fc-FcRn interface.

36. The polypeptide of any of paragraphs 1-35, comprising a mutation ina residue in a surface region that interacts with FcRn.

37. The polypeptide of paragraph 36, wherein the mutation is in aresidue chosen from: L251, I253, R255, P257, H285, N286, K288, T307,V308, L309, Q311, L314, H310, H433, N434, H435, or Y436.

38. The polypeptide of any of paragraphs 1-37, comprising a plurality ofmutations in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or allof the residues chosen from L251, I253, R255, P257, H285, N286, K288,T307, V308, L309, Q311, L314, H310, H433, N434, H435, or Y436.

39. The polypeptide of any of paragraphs 1-38, comprising a mutation ina residue that is a peripheral residue along the Fc-FcRn interface.

40. The polypeptide of paragraph 39, wherein the mutation is in aresidue chosen from M252, T256, T307, L309, Q311, H433, N434, Y436,N286, or K288.

41. The polypeptide of any of paragraphs 1-40, comprising a plurality ofmutations in 2, 3, 4, 5, 6, 7, 8, 9, or all of the residues chosen fromM252, T256, T307, L309, Q311, H433, N434, Y436, N286, or K288.

42. The polypeptide of any of paragraphs 1-41, comprising a mutation isin a residue that is non-contact residue in Fc-FcRn binding.

43. The polypeptide of paragraph 42, wherein the mutation is in aresidue chosen from A287, V308, N315, L314, L432, H429, E430, or A431.

44. The polypeptide of any of paragraphs 1-43, comprising a plurality ofmutations in 2, 3, 4, 5, 6, 7, or all of the residues chosen from A287,V308, N315, L314, L432, H429, E430, or A431.

45. The polypeptide of any of paragraphs 1-44, comprising a mutation ina residue which is a helix contact reside that enhances theconformational dynamics of a helix comprising 1, 2, 3, 4, 5, or all ofP247, K248, D249, T250, L251, or M252.

46. The polypeptide of paragraph 45, wherein the mutation is in aresidue chosen from P244, P245, T250, L251, P247, E380, M428, A378,D376, P257, V308, A287, L306, or H427.

47. The polypeptide of any of paragraphs 1-46, comprising a plurality ofmutations in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all of theresidues chosen from P244, P245, T250, L251, P247, E380, M428, A378,D376, P257, V308, A287, L306, or H427.

48. The polypeptide of any of paragraphs 1-47, comprising a mutationwhich is the introduction of a histidine along the Fc-FcRn interface.

49. The polypeptide of any of paragraphs 1-48, comprising 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more mutations, or one or more combination ofmutations, as described in Table 1.

50. The polypeptide of any of paragraphs 1-49, wherein the mutation isother than M252Y, S254T, T256E, L309N, T250Q, M428L, N434S, N434A,T307A, E380A, N434A, M252Y, S254T, T256E, or a combination thereof.

51. The polypeptide of any of paragraphs 1-50, wherein the mutation isin a residue other than residue M252Y, S254T, T256E, L309N, T250, M428,N434, N434, T307, E380, N434, M252, 5254, T256, or a combinationthereof.

52. The polypeptide of any of paragraphs 1-51, which does not have 1, 2,3, 4, 5, 6, 7, 8, 9, or all of the following mutation or mutations: (i)M252Y, S254T, and T256E; (ii) L309N; (iii) T250Q and M428L; (iv) M428Land N434A; (v) N434A; (vi) T307A, E380A, and N434A; (vii) M252W; (viii)V308F; (ix) V308F and N434Y; or (x) H435A.

53. The polypeptide of any of paragraphs 1-52, comprising a firstmutation chosen from M252Y, S254T, T256E, L309N, T250Q, M428L, N434S,N434A, T307A, E380A, N434A, M252Y, S254T, or T256E, and a secondmutation chosen from a mutation in Table 1 other than M252Y, S254T,T256E, L309N, T250Q, M428L, N434S, N434A, T307A, E380A, N434A, M252Y,S254T, and T256E.

54. The polypeptide of any of paragraphs 1-53, further comprising amutation in the Fc region that increases an effector function.

55. The polypeptide of paragraph 54, wherein the mutation is in aresidue chosen from S239, A330, I332, F243, G236, or a combinationthereof.

56. The polypeptide of any of paragraphs 1-55, further comprising amutation in the Fc region that decreases an effector function.

57. The polypeptide of paragraph 56, wherein the mutation is in aresidue chosen from K322, L234, L235, P331, N297, or a combinationthereof.

58. The polypeptide of any of paragraphs 1-57, wherein the Fc regioncomprises:

(a) 1, 2, 3, 4, 5, or all of the combinations of mutations chosen from:T256D/Q311V/A378V, H285N/T307Q/N315D, H285D/T307Q/A378V,T307Q/Q311V/A378V, T256D/N286D/T307R/Q311V/A378V, or T256D/T307R/Q311V;

(b) a mutation or a combination of mutations capable of disrupting an Fceffector function, or

(c) both (a) and (b).

59. The polypeptide of any of paragraphs 1-58, which comprises mutationsin residues chosen from: (i) T256, Q311, and A378; (ii) H285, T307, andN315; (iii) H285, T307, and A378; (iv) T307, Q311, and A378; (v) T256,N286, T307, Q311, and A378; or (vi) T256, H285, T307, Q311, and A378.

60. The polypeptide of any of paragraphs 1-59, which comprises mutationschosen from: (i) T256D, Q311V, and A378V; (ii) H285N, T307Q, and N315D;(iii) H285D, T307Q, and A378V; (iv) T307Q, Q311V, and A378V; (v) T256D,N286D, T307R, Q311V, and A378V; or (vi) T256D, H285D, T307R, Q311V, andA378V.

61. The polypeptide of any of paragraphs 1-60, further comprising amutation in a region other than the Fc region.

62. The polypeptide of any of paragraphs 1-61, comprising a plurality ofmutations, wherein at least one mutation is a compensating or beneficialmutation.

63. The polypeptide of any of paragraphs 1-62, which is an isolatedpolypeptide.

64. The polypeptide of any of paragraphs 1-62, which is a syntheticpolypeptide.

65. A composition comprising a polypeptide of any of paragraphs 1-64.

66. The composition of paragraph 65, further comprising a pharmaceuticalacceptable carrier.

67. A nucleic acid molecule encoding a polypeptide of any of paragraphs1-64.

68. A vector comprising a nucleic acid molecule of paragraph 67.

69. A cell comprising a nucleic acid molecule of paragraph 67 or avector of paragraph 68.

70. A kit comprising a polypeptide of any of paragraphs 1-64 andinstructions to use of the polypeptide.

71. A container comprising a polypeptide of any of paragraphs 1-64.

72. A method of producing a polypeptide, the method comprising culturinga cell of paragraph 69 under conditions that allow production of anantibody molecule, thereby producing the polypeptide.

73. The method of paragraph 72, further comprising isolating orpurifying the polypeptide.

74. A method of treating a disorder, the method comprising administeringto a subject in need thereof an effective amount of a polypeptide of anyof paragraphs 1-64 or a composition of paragraph 65 or 66, therebytreating the disorder.

75. A polypeptide of any of paragraphs 1-64, or a composition ofparagraph 65 or 66, for use in treating a disorder in a subject.

76. Use of a polypeptide of any of paragraphs 1-64, or a composition ofparagraph 65 or 66, in the manufacture of a medicament for the treatmentof a disorder in a subject.

77. A method of detecting a molecule, the method comprising contacting acell or a sample from a subject with a polypeptide of any of paragraphs1-64, thereby detecting the molecule.

EXAMPLES

A structure and network based framework was developed to interrogate theengagement of IgG with multiple Fc receptors (e.g., FcRn, C1q, TRIM21,FcγRI, FcγRIIa/b, and FcγRIIIa). Using this framework, features thatgovern Fc-FcRn interaction and multiple distinct pathways for enhancingFcRn binding in a pH specific manner were identified. Network analysisprovided a lens to study the allosteric impact of FcRn optimizationmutations on the distal FcγR engagement. Applying these principles, apanel of distinct Fc variants that enhance FcRn binding with robustbiophysical properties and wild-type like binding to activatingreceptors were engineered. Control polypeptides with these Fc variantshave shown a half-life improvement of >9 fold and robust effectorfunctions such as ADCC, ADCP, CDC and ADIN mediated by TRIM21.

Example 1: Structure Characterization and Molecular Features of Fc-FcRnInteraction

A co-crystal structure of the human FcRn protein in complex with the Fcdomain of a human IgG1 and human serum albumin was recently reported(Oganesyan et al., J Biol Chem, 2014. 289 (11): 7812-2). Inspection ofthe structure (FIG. 20A) shows that both the alpha and beta subunits ofthe FcRn molecule participate in binding to the Fc-domain, makingcontact with both CH2 and CH3 domains of the Fc. The primary interactionis mediated by the alpha-subunit on the FcRn side and CH2 domain on theFc side. The pH specific nature of binding is driven by the histidineresidues at positions 310 and 435 (Kabat numbering) which undergoprotonation at acidic pH and make critical contacts with FcRn glutamateat position 115 and aspartate at position 130. Mutation of either of thetwo histidine residues significantly reduces the binding affinity of Fcwith FcRn (Oganesyan et al., J Biol Chem, 2014. 289(43): 29874-80;Raghavan et al., Biochemistry, 1995. 34(45): 14649-57). In addition tothe H310 and H435, a number of other Fc residues (L251, 1253, R255,P257, H285, N286, K288, T307, V308, L309, Q311, L314, H433, N434, andY436) are involved in making molecular contacts with FcRn.

The structure of Fc has been solved at different pHs (Crispin et al.,Proc Natl Acad Sci USA, 2013. 110(38): E3544-6; Ahmed et al., J MolBiol, 2014. 426(18): 3166-79; Chen et al., ACS Chem Biol, 2016. 11(7):1852-61). Superposition of the crystal structure of Fc at pH 4.0 (pdb id4BYH) and 6.5 (pdb id 4Q7D) reveals a number of subtle changes,including lateral displacement of the 250-helix and differences in therelative orientation of CH2 (FIG. 20B). Given that FcRn binding aminoacid residues such as M252 and I253 are located on the 250-helix, theobserved displacement of the helix is expected to influence the FcRnbinding (Oganesyan et al., J Biol Chem, 2014. 289 (11): 7812-2).Further, the difference in the relative orientation of CH2 with respectto CH3 highlights the conformational flexibility of the CH2 region (FIG.20B) (Frank et al., J Mol Biol, 2014. 426(8): 1799-811). Given that theFcRn-Fc interface is found across both the CH2/CH3 domains of the Fc,the conformation dynamics of the CH2 domain is also expected toinfluence the FcRn binding. Deuterium exchange studies on the Fcresidues in the presence and absence of FcRn at acidic and neutral pHsshowed that FcRn binding to Fc molecule offers protection to its bindingFc residues; however, in the absence of FcRn molecule the 250-helixresidues at acidic pH showed enhanced Deuterium exchange suggesting thatpH change induces conformational change for this region (Walters et al.,J Biol Chem, 2016. 291(4): 1817-25; Jensen et al., Mol Cell Proteomics,2017. 16(3): 451-456).

Analysis of the strength of engagement of Fc with FcRn at acidic pHreveals that human FcRn binds to human Fc domain with weak affinity(>600 nM). The kinetic parameters further show that while the rate ofassociation (k_(on ˜)10⁵ M⁻¹s⁻¹) of Fc-FcRn interaction is comparable toa typical antibody-antigen interaction, the lower binding affinity isprimarily due to the fast dissociation rate (k_(off ˜)0.1 s⁻¹) ofFc-FcRn interaction (Suzuki et al., J Immunol, 2010. 184(4): 1968-76).The conformational flexibility of the CH2 subdomain is thought tocontribute to the poor k_(off) of FcRn binding. Improving the off rateof Fc-FcRn interaction can serve as a key facet for improved half-life(Datta-Mannan et al., J Biol Chem, 2007. 282(3): 1709-17).

Towards that, four sets of Fc residues, mutations to which could impingeon FcRn interaction and dissociation rate (k_(off)), were identified(FIG. 21A). This includes residues that make direct contact with FcRn aswell as peripheral and non-surface exposed residues that have thepotential to modify the interaction surface and residues that have thepotential to influence 250-helix dynamics. Indeed, some of thesepositions have been previously interrogated and half-life enhancingmutants such as M252Y/S254T/T256E (YTE), M428L/N434S (LS),T307A/E380A/N434A (AAA), T250Q/M428L (QL), V308P, include combinationsof mutations at the listed sites.

In the analysis, the role of each residue was first investigated insilico, and the network of interactions mediated by the residue wasidentified (FIG. 21B). Specific set of mutations were designed toenhance hydrophobic interactions in the Fc-FcRn interface and to enhancepolar and electrostatic interactions at the periphery of the Fc-FcRninterface. Mutations of non-contacting residues near the interface canenhance the electrostatic charge complementarity and the affinity byreducing the k_(off) rate (Lee and Tidor, Protein Sci, 2001. 10(2):362-77; Whitehead et al., Nat Biotechnol, 2012. 30(6): 543-8). The FcRnbinding site on Fc has overlaps with the binding site of intracellularreceptor TRIM21, protein A used for antibody purification and the Fc-Fcinteraction interface formed during hexamerization of IgG for CDCactivity. Substitution of at each position was assessed for impact onthese binding to TRIM21, protein A and Fc. More than 30 distinctpositions were chosen for substitutions. Combination of the individualmutations were designed based on the following guiding principles (a)avoiding introduction of large clusters of charge or hydrophobicity tominimize binding to solvent ions and impact on thermal stability of theIgG and (b) incorporate diversity in the types of interaction, not justelectrostatic or hydrophobic (c) include mutations across CH2 and CH3domains. Based on these considerations, more than 150 unique mutantcombinations were designed and experimentally evaluated.

IgG1 s incorporating the designed Fc variants on either actoxumab ormotavizumab Fab were recombinantly expressed and evaluated for bindingto human FcRn by biolayer interferometry using two different protocols:FcRn on NiNTA biosensor with IgG as the analyte, and IgG on an anti-CH1biosensor with FcRn as the analyte. The assays were used to quantifybinding at pH 6.0 followed by dissociation at pH 6.0 and pH 7.4, as wellas binding and dissociation at pH 7.4 (FIG. 11A). Fc variantsincorporating mutations at P257 and V308 had high affinity at pH 6.0 butdisplayed markedly slower off-rates at pH 7.4 and were not consideredfor further analyses. More than 10 distinct variants had greater than5-fold decrease in K_(d) as compared to IgG with WT Fc. Further, thesevariants decreased the k_(off) by more than 2.5-fold (FIG. 22).

The FcRn binding site on the Fc domain considerably overlaps with theprotein A and Trim21 binding sites as determined from their complexcrystal structures (FIG. 27A). The Fc domain also binds to C1q tomediate CDC, however the C1q binding site does note overlap with FcRnbinding site on Fc. C1q is naturally found as hexamer and a homo-hexamerassembly of Fc domains is expected to not only have a better C1q bindingand also CDC. The Fc-Fc interface for formation of homo-hexamer overlapswith the FcRn binding site (FIG. 27A). Superposition of crystalstructures of Fc domain shows that the orientation of the CH2 domain inthese structures varies with respect to the CH3 domain illustrating theconformational flexibility of the CH2 domain (FIG. 27B). Since the FcRnbinding site is at the interface across both the CH2 and CH3 domainstherefore this conformational flexibility may be important for itsbinding.

Example 2: Effect of Fc Region Mutations on pH Specific Fc-FcRn Binding

The FcRn binding affinity of Fc variants FcMut008 and FcMut015 wasassessed by Octet. Antibody was immobilized on the Octet tip by anti-CH1antibody and FcRn was in solution. As shown in FIG. 6, FcMut008 andFcMut015 had increased affinity to FcRn. The binding is pH specific andincreased binding at pH 6.0 was observed.

Example 3: Cell-Based FcRn Binding Competition Assay

Cell-based competition assay provides a robust, specific, linear assayto show differences in relative binding of Fc variants. FcRn-expressingcells were obtained by transient transfection of FcRn alpha and β2m.Cells were incubated at pH6.0 with dilutions of IgG and fixedconcentration of fluorescently-labeled Fc (Fc-A488). Cell-boundfluorescence was read by FACS. The results are shown in FIG. 7. FcMut008and FcMut015 showed improved FcRn binding at pH6.0.

Example 4: Effect of Fc Region Mutations on FcγR Engagement

The binding of exemplary mutant antibody molecules to FcγRI and FcγRIIIawas determined. As shown in FIG. 8, FcMut008 and FcMut015 retained andin some instance enhanced FcγR engagement.

Example 5: Effect of Fc Region Mutations on Thermal Stability andBiophysical Characterization of Fc Variants

The thermal stability of exemplary mutant antibody molecules wasdetermined. The thermal stability was measured by SYPRO orange. As shownin FIG. 9, FcMut008 and FcMut015 retained high melting temperature.

The impact of incorporating exemplary Fc variants on biophysicalattributes was experimentally assessed. IgGs incorporating the Fcvariants on motavizumab Fab were tested on SE-HPLC. All samples elutedat similar retention times as wild-type Fc, and displayed cleanmonomeric profile, and no aggregates were detected (FIG. 23). The IgGswere also assessed for the thermal stability of the CH2 and CH3 domainsby Differential Scanning Fluorimetry (DSF). The melting temperature(T_(m)) of the wild type human CH2 and CH3 domain, as measureddifferential scanning calorimetry, is approximately 70° C. and 81.5° C.,respectively (Ionescu et al., J Pharm Sci, 2008. 97(4): 1414-26). TheDSF experimental results in this Example yielded similar results with aCH2 and CH3 T_(M) of 68.8° C. and 80.8° C., respectively. The half-lifeextending Fc variant YTE has been reported to decrease the T_(M) of theCH2 domain by 6.7° C. (Majumdar et al., MAbs, 2015. 7(1): p. 84-95.). Inthe experiments described in this Example, the T_(M) of the CH2 domainof YTE was 7.2° C. lower than WT. Additionally, mutations at 247, 257,and 308 significantly impacted the T_(M) of CH2. The exemplary Fcvariants (FcMut183, FcMut197, FcMut213, FcMut215, FcMut228, FcMut229)were thermally stable with the T_(M) of the CH2 domain>64° C. (FIG. 23).

Example 6: Evaluation of Fc Variants in Transgenic Mice Model

Tg32 mice were homozygous, 8 week old, males. There were 4 mice per testarticle group. The test articles included CDA1-WT, CDA1-FcMut008, andCDA1-FcMut015. The mice were dosed at 10 mg/Kg by IV administration.Data were collected at thirteen time points (1 h, 8 h, 1 d, 2 d, 3 d, 4d, 6 d, 8 d, 10 d, 13 d, 16 d, 19 d, and 22 d). Human IgG was quantifiedby ELISA using an anti-hIgG polyclonal antibody.

Tg32 is a human FcRn transgenic mouse model that can be used in drugdiscovery for early assessment and prediction of human pharmacokineticsof monoclonal antibodies. Monoclonal antibody clearance in Tg32homozygous mice has the strongest correlation to monoclonal antibodyclearance in humans (Avery et al. MAbs. 2016; 8(6):1064-78).

CDA1 (actoxumab) is known to have a half-life of >25 days in human Invivo evaluation with additional mAbs in Tg32 model was performed. Thedifferent constructs can also be evaluated on Tg276 mice which arereported to have increased half-life differences between IgG variants.The results are shown in Table 2 and FIG. 10. FcMut015 increased thehalf-life of CDA1 in Tg32 mice.

TABLE 2 Half-Lives of Exemplary Antibody Molecules in Tg32 HomozygousMice t_(1/2) C_(max) C_(last) AUC_(inf) Group (hr) (ug/ml) (ug/ml)(hr*ug/ml) Rsq WT 261.17 116.03 15.40 24108.03 0.99 FcMut008 231.92131.33 15.74 25687.39 0.99 FCMut015 436.69 151.82 27.69 42735.9 0.93

Example 7: Fc Engineering of Exemplary Antibodies

FcRn interaction with IgG is believed to be mediated through Fc. Thebinding of Fc to FcRn is generally pH specific (typically little or nobinding at pH7.4 and strong binding in acidic environment). Structure ofFcRn in complex with Fc domain of IgG1 is known and each FcRn moleculebinds to an Fc-monomer. Fab domains can also influence binding of IgG toFcRn.

Network analysis of Fc-FcRn complex highlights the centrality of H310 inengagement with FcRn. H310 is highly interconnected to multiple otherhighly networked residues. Mutations in the H310 cluster, andneighboring (connected nodes) may strengthen the H310 network. Analysisof sub-networks informs introduction of synergistic mutations forfavorable FcRn interaction, with minimal impact on other Fc residues.

To identify Fc variants with improved binding to FcRn at pH 6.0, variousFc mutations were engineered into IgG1 Motazivumab (WT). All antibodieswere assessed for binding to FcRn using biolayer interferometry.Briefly, anti-CH1 biosensors (ForteBio) were loaded with each antibodyof interest. Loaded biosensors were exposed to recombinant FcRn proteinat pH 6.0 to detect binding. After saturation, biosensors were exposedto buffer alone at pH 6.0 to measure dissociation of the FcRn from eachantibody. The result is a response curve representing on rate and offrate of the antibody. These rates were calculated using the ForteBiosoftware and compared to the rate of wild type Motavizumab. The foldincrease in on-rate and the fold decrease in off-rate as compared towild type are listed in the table. Numerous antibody mutationssignificantly increased and decreased on and off rate, respectively(FIG. 11A). Also shown is the binding of a representative Fc variant toFcRn at a pH range of 6.0 to 7.4. It is important that the affinity ofthe Fc mutant antibodies for FcRn is improved at pH 6.0 but notsignificantly enhanced at a higher pH such as pH 7.4. FIG. 11Bdemonstrates that this representative antibody still shows poor bindingto FcRn at pH 7.4, a desirable feature.

FIG. 11C shows the interaction of the CH2 domain of the antibody Fc withthe FcRn molecule. The engineering efforts described herein attempted toimprove shape complementarity (SC), electrostatic charge complementarity(CC), and hydrophobic complementarity (HC). A few positions on the Fcare noted as important for the interaction. Also noted is the 250 helix.This helix is dynamic and moves depending on the pH of the environment.This is important in the binding of the Fc domain to FcRn at pH 6.0 butnot pH 7.4.

Exemplary Fc variants were tested in developability assays, the resultsof which are summarized in FIG. 12. Assays, including Sypro orange, SDSPAGE and SEC-HPLC, were performed. For expression determination,constructs were transfected into Expi293 cells in 96 well culture dishesusing ExpiFectamine as described by the manufacturer. After 5-7 days,supernatants were quantified using biolayer interferometry (Octet)equipped with anti-human CH1 biosensors using a motavizumab standardcurve. For protein A binding assessment the following was performed.Constructs were transfected into Expi293 cells in 30 mL cultures usingExpiFectamine as described by the manufacturer. After 5-7 days,supernatants were harvested and antibodies purified. Using biolayerinterferometry with protein A biosensors, Fc-modified antibody affinityto protein A was measured and compared to antibody containing a wildtype Fc domain.

All Fc variants, expressed in the context of the Motavizumab antibody,were separated by SDS-PAGE under reducing and non-reducing conditions.Briefly, 2 μg of antibody in 5 μL of water was mixed with 5 μL ofLaemmli Sample Buffer (BioRad Catalog #161-0737), with and ofβ-mercaptoethanol (BioRad Catalog #161-0710). The samples were boiled at95° C. for ten minutes and then briefly centrifuged. Samples were thenrun on a 4-12% Bis-Tris NuPAGE gel (Thermo Scientific # NP0321BOX) in1×MES running buffer in an XCell SureLock Gel Electrophoresis Cell(Novex Catalog #090403-839) alongside the SeeBlue Plus2 molecular weightstandard (Invitrogen Catalog # LC5925). The samples were run at 200V for35 minutes. The gels were stained with SimplyBlue SafeStain (Novex #LC6065) following manufacturer's protocol and imaged on the BioRadChemiDoc MP Imaging System.

HPLC based size exclusion chromatography (HPLC-SEC) is an analyticaltool used to determine the apparent size of a protein, monomeric purity,and the apparent level non-specific column adsorption between theprotein and the silica based sizing resin. The impact of Fc mutations onthe IgG elution profile was assessed on a Phenomenex Biosep 3000scolumn. Briefly the IgGs with various Fc variants were diluted to 1mg/ml in PBS pH 7.4 and 20 tit was injected into the column. The elutiontime and percentage purity was recorded.

The thermal stability of the mAbs was determined by differentialscanning fluorescence (DSF). DSF monitors the conformational stabilityof a protein as it is exposed to increasing thermal stress. The dye,SYPRO ORANGE®, fluoresces in a hydrophobic environment, such ashydrophobic core residues that are exposed during thermally triggeredprotein unfolding, or denaturation. By monitoring the fluorescentsignal, the unfolding of CH2, CH3 and Fab can be monitored. Various Fcvariants with Motavizumab Fab were evaluated in a SYPRO orange assay.Briefly 15 μL of IgG at 0.5 mg/mL was mixed with 15 μL of 1:500 dilutedSYPRO Orange dye and assessed by a thermocycler with fluorescent readcapabilities using a 1° C. ramp from 40° C. to 99° C. The midpointbetween native state and first unfolding event was reported as thetransition temperature or melt temperature (Tm).

All Fc mutations were introduced in IgG1 (m3 allotype) heavy chain geneand cloned into pcDNA3.1(C). The light chain genes were cloned intopcDNA3.1(A). In all cases, the native signal peptide was replaced withan osteonectin signal peptide (GenBank accession # AAA60993).Co-expression of the heavy and light chain vectors was performed bytransient transfection in Expi293 cells using the Expi293 transfectionkit (Thermo Fisher catalogue # A14524) following the manufacturer'sprotocol. The heavy and light chain vectors were co-transfected at a 1:2ratio. Cell culture supernatant was harvested 5 to 7 days posttransfection and purified on the AKTA 10 FPLC system (GE) using HiTrapMabSelect SuRe protein A columns (GE) following manufacturer'sinstructions.

All antibodies were purified from cell culture supernatant using 1 mLcolumns packed with mAb select sure protein A resin (GE catalogue#17543801) using the AKTA purifier 10 FPLC system. Briefly, sterilefiltered cell culture supernatant was loaded onto the columns at a flowrate of 2 mL/minute. Columns were washed with 10 column volumes of PBSN(1×PBS with 0.05% sodium azide). Antibodies were eluted with 10 columnvolumes of elution buffer (100 mM glycine pH 2.5) and neutralized byaddition 17.5% v/v of neutralization buffer (1M Tris, 1M NaCl, pH 8.0)and collated in 1 mL fractions. The chromatogram for absorbance at 280nm was used to identify elution fractions containing the antibody. Allantibodies were then dialyzed into 1×PBS using 10,000 dalton molecularweight cut-off cassette (Thermo Fisher catalogue #66380).

Protein A binding was functionally determined by the ability of allantibodies to be purified by FPLC using protein A columns. Afterpurification, protein A binding was further assessed by quantifying aknown amount of antibody using the Octet QKe system and protein Abiosensors (Pall catalogue #18-5012) following the standard quantitationprotocol provided with the Octet data acquisition software.

Expi293 cells were co-transfected with a plasmid encoding the humanα-FcRn with a 6× histidine tag on the C-terminus and a plasmid encodinghuman 132M. Cell culture supernatant was harvested 4 days posttransfection. FcRn was purified from cell culture supernatant using theAKTA pure FPLC system with a HisTrap HP column (GE catalogue#17-5247-01). Post purification the protein was dialyzed into 1×PBS pH6.0.

Screening assays were performed using anti-CH1 Fab biosensors on theOctet QKe system. Briefly, purified IgG at 10 μg/mL is loaded onto ananti-CH1 biosensor for 180 seconds. After a 60 second baseline step in1×PBS pH 6.0, the IgG loaded tip is exposed to FcRn at a concentrationof 50 μg/mL for 60 seconds, followed by dissociation for 60 seconds inPBS pH 6.0, and an additional 30 seconds in PBS pH 7.4. In addition,FcRn binding was performed using NiNTA biosensors. Briefly, recombinanthuman FcRn at 5 μg/mL is loaded onto a NiNTA biosensor for 180 seconds.After a 60 second baseline step in 1×PBS pH 6.0, the FcRn loaded tip isexposed to IgG at a concentration of 250 nM (37.5 μg/mL) for 60 seconds,followed by dissociation for 60 seconds in PBS pH 6.0, and an additional30 seconds in PBS pH 7.4. After assay completion of each assay, aquantitative assessment of the affinity constant (K_(D)) at pH 6.0 isperformed using the ForteBio octet software and a qualitative assessmentis performed by plotting the response rate over time, allowing forvisualization of the association of IgG to FcRn at pH 6.0 and thesubsequent dissociation at pH 6.0 and pH 7.4.

As shown in FIG. 12, all Fc variants performed comparably to the WTantibody in all of these experiments.

Example 8: In Vivo Assessment of Half-Life and Pharmacokinetic Analysisof Engineered Antibodies

Motavizumab wildtype was compared to Motavizumab containing three of Fcengineering mutations in the in vivo assessment of engineered antibodyhalf-life. Antibodies (2-5 mg/kg) were administered to mice transgenicfor human FcRn and daily samples of mouse serum were obtained (day 0-day4). ELISA was performed on serum to quantify the amount of Motazivumabin the serum.

The amount of human IgG present in mouse serum was determined using ahuman IgG quantitation ELISA kit (Bethyl Labs catalogue # E80-104)following the manufacturer's protocol. All serum samples were titratedin a twofold serial dilution starting at 1:50 dilution and ending at a1:6400 dilution. Each ELISA plate included a human reference standardcurve provided with the kit in duplicate or triplicate. The standardcurve contains the following concentrations: 500.0, 250.0, 125.0, 62.5,31.3, 15.6, 7.8, and 3.9 ng/mL. The lower limit of detection wasconsidered to be the A450 nm value obtained for the second to last pointon the reference standard, which was 7.8 ng/mL. Because the startingdilution of the serum was 1:50, this puts the level of detection theassay at ˜0.4 μg/mL (7.812 ng/mL×50 fold dilution). The followingprocedures were followed to calculate the IgG levels: (1) perform a fourparameter logistic regression (4PL) on the standard curve; (2) set themaximum acceptable A450 nm signal as the reading for the third titrationpoint on the standard curve; (3) set the minimum acceptable A450 nmsignal as the reading for the seventh titration point on the standardcurve; (4) mask all samples titration points that fall above the maximumacceptable signal and below the minimum acceptable signal; (5) user thereference standard 4PL curve fit to calculate the concentration for eachtitration point with an acceptable A450 signal and multiply that valueby the dilution factor of that titration point; (6) for each sampletitration, calculate the mean value for the calculated concentrationacross titration series.

ELISA results were converted to percent of antibody remaining based onthe day 0 timepoint representing 100%. As shown in FIGS. 13A-13B, allthree antibody variants demonstrated extended half-life in thiswell-established mouse model of antibody half-life. Motavizumabwild-type has a half-life of 32 hours. FcMut043 and FcMut045 mutantsbuilt on FcMut008 show significant half-life improvement. FcMut045mutant enhanced half-life 5.2 fold (about 166 hours half-life). Thehalf-life as well as the other parameters in FIG. 13B were calculatedusing the Winonlin software.

Similar experiments were conducted with later stage Fc variants in thecontext of Motazivumab. When the Motavizumab variants were administeredat a dose of 5 mg/kg, next generation variants (FcMut171, FcMut183,FcMut186, and FcMut197) demonstrated further enhanced half-life withmore than nine fold increase in half-life observed with FcMut213 (FIGS.14A-14B). One of the early variants (FcMut045) was included todemonstrate the improved half-life seen with the later stage designs ascompared to the early stage designs. Similar results were observed whenthe Motavizumab variants were administered at a dose of 2 mg/kg (FIGS.14C-14D).

To evaluate if the enhanced binding of Fc variants to human FcRntranslated to increased serum persistence and longer circulatinghalf-life life, a pharmacokinetic study of IgGs containing the differentFc variants was performed in Tg276 transgenic mice. Transgenic mousemodels expressing human FcRn have been developed by the Jacksonlaboratory (JAX) to study PK of human Fc-containing biotherapeutics. TheTg276 mice are null for mouse FcRn alpha chain and express the humanFcRn alpha transgene under the control of a constitutive promoter(actin) and use the mouse β2microglobulin. The Tg276 homozygous orhemizygous mice have been widely used to differentiate half-lives ofantibody variants.

For the in vivo study, Tg276hemi FcRn transgenic mice were dosed with 2or 5 mg/kg of mAb intravenously. Each mAb group had 4 mice/group. Bloodwas collected at several time points between 1 hour and 21 days, and IgGtiters were determined by quantitative ELISA as described herein. PKparameters were determined for each group of mice with anon-compartmental model using Phoenix WinNonlin version 7.0 (Certera).

The results are shown in FIGS. 14A-14D.

Example 9: In Vivo Assessment of Half-Life of Engineered Antibodies

Two Zika antibodies (ZVA and ZVB) as well as wild type Motavizumab (MVZ)were administered to mice transgenic for human FcRn at a dose of 2-5mg/kg, and daily samples of mouse serum were obtained (day 0-day 4).ELISA was performed on serum to quantify the amount of Motavizumab.ELISA results were converted to percent of antibody remaining based onthe day 0 timepoint representing 100%. ZVA antibody represents the Aseries antibody A-3/2. The ZVB antibody represents the A series antibodyA-5/1 containing the affinity enhancing light chain modification S92Y.As shown in FIG. 15, ZVB had a much longer half-life than eitherMotavizumab or ZVA (both ZVA and ZVB had longer half-life thanMotavizumab). These antibodies contained wild-type Fc regions so theextended half-life of the ZVB antibody is a property of the antibodyitself, and not any Fc engineering.

In the next experiment, Motavizumab wild type (MVZ WT) was testedagainst the ZVB antibody (ZB-1/4, ZKB in the graph) and the ZVB antibodycontaining Fc modification 156 (L234A/L235A (“LALA”) andT256D/T307R/Q311V (half-life extension), ZKB-156 in the graph) as wellas the “LS” half-life extension Fc mutation (ZKB-LS). Both LS and Fcmodification 156 extended half-life significantly compared to thewild-type ZVB (FIG. 16). These data demonstrate that the Visterramutation is comparable to the literature derived LS mutation, and alsodemonstrates that the FC engineering efforts described herein can extendthe half-life of an antibody that already has a very long half-life inthis transgenic mouse model. “ZVB” is indicated as “ZKB” in FIG. 16.

Example 10: Binding to Fc Receptors and Impact on Effector Functions

Exemplary Fc variants were assessed on multiple assays for FcγRI,FcγRIIA (mediator of opsonophagocytosis), FcγRIIB, FcγRIIIA (mediator ofADCC), and C1q (mediator of CDC).

Binding to Fcγ receptors I, IIa, IIb, IIIa, and IIIa V176F (R&D Systemscatalogue #1257-FC-050, 1330-CD-050, 1875-CD-050, 4325-FC-050, and8894-FC-050) was measured by ELISA. All Fc variants were tested in thecontext of the Motavizumab, Rituximab, or Actoxumab antibody. Briefly,Fc receptors were coated on an ELISA plate (VWR catalogue #62409-002) at1 μg/mL (0.1 μg/well) in PBS and stored at 4° C. overnight. Plates werewashed three times with PBST (1×PBS 0.05% Tween20). Antibodies weretitrated threefold in PBST-BSA from 100 μg/mL to 0.05 μg/mL and 100 μLwas added to each well of the ELISA plate and incubated for 1 hour atroom temperature. Plates were washed three times with PBST. Goatanti-human Fc (Jackson catalogue #109-035-098) was diluted 1:5000 inPBST-BSA and 100 μL was added to each well and incubated for 1 hour at 4C. Plates were washed six times with PBST. Plates were developed usingthe TMB Microwell Peroxidase Substrate Kit (VWR catalogue #95059-156).The reaction was stopped after 10 minutes by the addition of 1N sulfuricacid and absorbance at 450 nm was measured. The values of antibodyconcentration (x-axis) and absorbance at 450 nm (y-axis) were fit to afour parameter logistic regression (4PL) curve. The curve fit was thenused to determine the EC50 (the midpoint of the 4PL) for each Fcvariant.

Binding to Fcγ receptors IIa and IIb was measured by BioLayerInterferometry using the Octet QKe system. Briefly, FcγIIa and FcγIIb(R&D Systems catalogue #1330-CD-050 and 1875-CD-050) were diluted to 5μg/mL in PBS. The receptors were immobilized via a C-terminal 6×histidine tag (SEQ ID NO: 2) to Ni-NTA biosensors (Pall catalogue#18-5101) for 180 seconds followed by a 60 second baseline step in PBS.The biosensors were then exposed to the various Fc variants at aconcentration of 50 μg/mL in PBS for 120 seconds followed by adissociation step in PBS for an additional 120 seconds. The max bindingresponse during the association step for each variant was reported andcompared to the wild type Fc response.

Binding to C1q was measured by ELISA. All Fc variants were tested in thecontext of the Motavizumab antibody. Briefly, antibodies were coated onan ELISA plate (VWR catalogue #62409-002) at 25 μg/mL (2.5 μg/well) inPBS and stored at 4° C. overnight. Plates were washed three times withPBST (1×PBS 0.05% Tween 20). Purified C1q (Quidel Corporation catalogue# A400) was titrated threefold in PBST-BSA (1×PBS 0.05% Tween20 1% BSA)from 12.5 μg/mL to 0.02 μg/mL and incubated for 90 minutes at roomtemperature. Liquid was aspirated from wells and polyclonal rabbitanti-human C1q (Agilent catalogue # A013602-1) was diluted in PBST-BSAto a final concentration of 1 μg/mL and 100 μL was added to each welland incubated for 1 hour at room temperature. Plates were washed threetimes with PBST. Polyclonal swine anti-rabbit-HRP (Agilent catalogue #P021702-2) was diluted to 0.5 μg/mL in PBST-BSA and 100 μL was added toeach well and incubated for 1 hour at room temperature. Plates werewashed six times with PBST. Plates were developed using the TMBMicrowell Peroxidase Substrate Kit (VWR catalogue #95059-156). Thereaction was stopped after 10 minutes by the addition of 1N sulfuricacid and absorbance at 450 nm was measured. The values of antibodyconcentration (x-axis) and absorbance at 450 nm (y-axis) were fit to afour parameter logistic regression (4PL) curve. The curve fit was thenused to determine the EC50 (the midpoint of the 4PL) for each Fcvariant.

The results are shown in FIGS. 17A-17D.

Example 11: CDC Activity of Engineered Antibodies

Complement dependent cytotoxicity (CDC) activity of exemplary Fcvariants (Rituximab Fab) was examined.

CDC assays were performed using CD20+ Raji cells and low toxicity guineapig complement (Cedarlane Laboratories Product # CL4051). Complementinduced cell lysis was measured using the CYTOTOX 96® Non-RadioactiveCytotoxicity Assay from Promega (catalogue # G1780) following themanufacturer's protocol. All Fc variants were tested in the context ofthe anti-CD20 antibody, Rituximab. Briefly, antibody concentrations weretitrated fourfold ranging from 20 μg/mL to 0.005 μg/mL and incubatedwith 20,000 target cells per well at 37° C. for 30 minutes. Complementwas then added to the cells and incubated an additional 2 hours at 37°C. Additionally, cell lysis buffer provided with the CYTOTOX kit wasadded to control wells to measure the maximum cell lysis. A negativecontrol antibody, Motavizumab, was used to measure the background signalof an irrelevant antibody. The background signal and maximum lysissignal were used to calculate the percent of cell lysis for each Fcvariant. The values of antibody concentration (x-axis) and percent lysis(y-axis) were fit to a four parameter logistic regression curve. Thecurve fit was then used to determine the EC50 (concentration needed toobtain 50% lysis) and the maximum lysis for each Fc variant.

The results are shown in FIG. 18.

Example 12: ADCC Activity of Engineered Antibodies

Antibody dependent cellular cytotoxicity (ADCC) activity of exemplary Fcvariants (Rituximab Fab) was examined.

ADCC assays were performed using the ADCC Reporter Bioassay with CD20⁺WIL2-S target cells from Promega (catalogue # G7014) following themanufacturer's protocol. All Fc variants were tested in the context ofthe anti-CD20 antibody, Rituximab. Antibody concentrations were titratedfivefold ranging from 5 μg/mL to 0.0016 μg/mL. The values of antibodyconcentration (x-axis) and fold induction of the luminescent reportergene (y-axis) were fit to a four parameter logistic regression (4PL)curve. The curve fit was then used to determine the EC50 (the midpointof the 4PL) and the maximum induction for each Fc variant.

The results are shown in FIGS. 19A-19B. Exemplary Fc variants retain andin some cases enhance ADCC activity.

Example 13: ADIN Activity of Engineered Antibodies

TRIM21 is a cytosolic receptor that binds with Fc of IgG. TRIM21 plays arole in mediating intracellular recognition and neutralization of Fcbound viruses. TRIM21-mediated neutralization is known as antibodydependent intracellular neutralization (ADIN).

Binding to TRIM21 was measured by ELISA. All Fc variants were tested inthe context of the Motavizumab or Actoxumab antibody. Briefly,antibodies were coated on an ELISA plate (VWR catalogue #62409-002) at25 μg/mL (2.5 μg/well) in PBS and stored at 4° C. overnight. Plates werewashed three times with PBST (1×PBS 0.05% Tween20). TRIM21-GST(Antibodies Online catalogue # ABIN1323621) was titrated threefold inPBST-BSA (1×PBS 0.05% Tween20 1% BSA) from 12.5 μg/mL to 0.02 μg/mL andincubated for 90 minutes at room temperature. Liquid was aspirated fromwells and two rabbit anti-TRIM21 antibodies (AbCam catalogue # ab91423and ab96800) were mix together in PBST-BSA at a final concentration of 1μg/mL each and 100 μL was added to each well and incubated for 1 hour atroom temperature. Plates were washed three times with PBST. Polyclonalswine anti-rabbit-HRP (Agilent catalogue # P021702-2) was diluted to 0.5μg/mL in PBST-BSA and 100 μL was added to each well and incubated for 1hour at room temperature. Plates were washed six times with PBST. Plateswere developed using the TMB Microwell Peroxidase Substrate Kit (VWRcatalogue #95059-156). The reaction was stopped after 10 minutes by theaddition of 1N sulfuric acid and absorbance at 450 nm was measured. Thevalues of antibody concentration (x-axis) and absorbance at 450 nm(y-axis) were fit to a four parameter logistic regression (4PL) curve.The curve fit was then used to determine the EC50 (the midpoint of the4PL) for each Fc variant.

A TRIM21 binding ELISA was performed to evaluate if the Fc variantsimpacted binding to TRIM21. The results are shown in FIG. 26. Fcvariants FcMut045, FcMut183, FcMut197, FcMut213, FcMut215, FcMut228 hadTRIM21 binding EC50s that were within 1.5 fold of WT EC50.

Example 14: Enhancement of Mucosal Uptake by Fc Mutations

FcRn transports IgG across different cellular barriers such as themucosal epithelium lining the intestine and the alveolar surfaces.Modification of FcRn binding provides a mechanism to enhance mucosallocalization that confers immune protection. Exemplary Fc mutants areexpected to enhance mucosal uptake in a similar fashion

Example 15: Impact of FcRn Affinity Enhancing Mutations on Engagementwith Fc Receptors and Effector Functions

The impact of FcRn affinity enhancing mutations on binding of Fc toother receptors was experimentally evaluated. The Fc region of IgG iscapable of binding to many different Fc receptors and the mechanism ofaction for many therapeutic antibodies relies on engagement with Fcreceptors. Mutations, even at positions distant from the Fc receptorbinding site, can impact engagement of Fc with receptors such as FcγRI,FcγRIIa/b, FcγRIIIa, C1q, TRIM21. While Fc receptor TRIM21 binds to asite that overlaps with FcRn binding site on CH2-CH3, other receptorssuch Fcγ receptors and C1q engage at the interface formed by dimericCH2. As such any mutations introduced for enhancing half-life should beassessed for its impact on binding to other receptors. Mutationsintroduced to enhance half-life can The Fc variants (FcMut183, FcMut197,FcMut213, FcMut215, FcMut228, FcMut229, YTE, LS) were incorporated intorituximab Fab and together with WT rituximab, were evaluated for bindingto FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, C1q and TRIM21. As shown in FIG.24, introduction of Fc variants FcMut183, FcMut197, FcMut213, FcMut215,FcMut228 and FcMut229 did not significantly impact engagement with thetested Fc receptors.

While binding to Fc receptors is necessary for activities such as ADCC(FcγRIIIa), CDC (C1q) and ADIN (TRIM21), it may not be sufficient inmediating these activities. For example, a hexameric assembly of Fc isrequired to elicit CDC activity. The Fc hexamer assembly involvesformation of an Fc-Fc interface, and residues involved in formation ofthis interface partially overlaps with FcRn binding site. As such, itwas important to evaluate the impact of the Fc mutations on not justbinding to Fc receptors but also activities such as CDC and ADCC.Rituximab with various Fc variants (FcMut183, FcMut197, FcMut213,FcMut215, FcMut228, FcMut229, YTE, LS, WT) were evaluated for ADCC andCDC activities as described in the methods section. The resultsconfirmed that Fc variants FcMut183, FcMut197, FcMut213, FcMut215,FcMut228 and FcMut229 did not negatively impact ADCC activity and infact showed a slight enhancement in CDC activity. Rituximab containingYTE had no detectable CDC activity and significantly reduced ADCCactivity. Similarly another mutant FcMut197 had reduced ADCC and CDCactivity. Inspects of the network map of these mutations provides aninsight into the possible reason for loss of ADCC and CDC activity.

Example 16: Persistence of Optimized Fc Variants in Sera of TransgenicMice

To evaluate whether the enhanced binding of Fc variants to human FcRntranslated to increased serum persistence and longer circulatinghalf-life life, a pharmacokinetic study of IgGs containing the differentFc variants was performed in Tg276 transgenic mice. Transgenic mousemodels expressing human FcRn can be used to study PK of humanFc-containing biotherapeutics (Roopenian et al., Methods Mol Biol, 2010.602: 93-104; Petkova et al., Int Immunol, 2006. 18(12): 1759-69). TheTg276 mice are null for mouse FcRn alpha chain and express the humanFcRn alpha transgene under the control of a constitutive promoter(actin) and use the mouse β2microglobuli. The Tg276 homozygous andhemizygous mice can be used to differentiate half-lives of antibodyvariants. For the PK study, Tg276hemi FcRn transgenic mice wereintravenously administered with 5 mg/kg of IgG (motavizumab Fab on WT ormodified Fc). Retro-orbital blood collection was performed at severaltime points, and IgG titers were determined by quantitative ELISA. IgGscontaining the engineered Fc variants persisted in serum much longerthan wild-type Motavizumab (FIG. 25A). Pharmacokinetic parameters fromnon-compartmental analysis indicates Motavizumab with Fc variants havegreater than two fold slower clearance rate and significant increases inβ-phase elimination half-life and area under the curve (AUC)measurements (FIG. 25B).

INCORPORATION BY REFERENCE

All publications, patents, and Accession numbers mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

What is claimed is:
 1. A polypeptide comprising the CH2 and CH3 domainsof an Fc region, wherein the CH2 and CH3 domains of the Fc regioncomprises mutations from any of (i)-(vi): (i) T256D, Q311V, and A378V;(ii) H285N, T307Q, and N315D; (iii) H285D, T307Q, and A378V; (iv) T307Q,Q311V, and A378V; (v) T256D, N286D, T307R, Q311V, and A378V; or (vi)T256D, H285D, T307R, Q311V, and A378V.
 2. The polypeptide of claim 1,which is an isolated polypeptide or a synthetic polypeptide.
 3. Thepolypeptide of claim 1, wherein the CH2 and CH3 domains of the Fc regioncomprises the mutations T256D, Q311V, and A378V.
 4. The polypeptide ofclaim 1, wherein the CH2 and CH3 domains of the Fc region comprises themutations H285N, T307Q, and N315D.
 5. The polypeptide of claim 1,wherein the CH2 and CH3 domains of the Fc region comprises the mutationsH285D, T307Q, and A378V.
 6. The polypeptide of claim 1, wherein the CH2and CH3 domains of the Fc region comprises the mutations T307Q, Q311V,and A378V.
 7. The polypeptide of claim 1, wherein the CH2 and CH3domains of the Fc region comprises the mutations T256D, N286D, T307R,Q311V, and A378V.
 8. The polypeptide of claim 1, wherein the CH2 and CH3domains of the Fc region comprises the mutations T256D, H285D, T307R,Q311V, and A378V.
 9. The polypeptide of claim 1, which further comprisesa hinge region between the CH2 and CH3 domains of the Fc region.
 10. Thepolypeptide of claim 1, which is an antibody molecule.
 11. Thepolypeptide of claim 10, wherein the antibody molecule is a chimericantibody molecule or a murine antibody molecule.
 12. The polypeptide ofclaim 10, wherein the antibody molecule is a human antibody molecule ora humanized antibody molecule.
 13. The polypeptide of claim 1, whichfurther comprises a heavy chain immunoglobulin variable region, a lightchain immunoglobulin variable region, or both.
 14. The polypeptide ofclaim 1, which is an immunoglobulin chain or a fragment thereof.
 15. Thepolypeptide of claim 1, which is a fusion protein.
 16. A compositioncomprising the polypeptide of claim
 1. 17. The composition of claim 16,further comprising a pharmaceutically acceptable carrier.
 18. A nucleicacid molecule encoding the polypeptide of claim
 1. 19. A vectorcomprising the nucleic acid molecule of claim
 18. 20. A cell comprisingthe nucleic acid molecule of claim
 18. 21. A kit comprising thepolypeptide of claim 1 and instructions to use of the polypeptide.
 22. Acontainer comprising the polypeptide of claim
 1. 23. A method ofproducing a polypeptide, the method comprising culturing the cell ofclaim 20 under conditions that allow production of an antibody molecule,thereby producing the polypeptide.
 24. The method of claim 23, furthercomprising isolating or purifying the polypeptide.
 25. A method oftreating a disorder, the method comprising administering to a subject inneed thereof an effective amount of the polypeptide of claim 1, therebytreating the disorder.
 26. A method of detecting a molecule, the methodcomprising contacting a cell or a sample from a subject with thepolypeptide of claim 1, thereby detecting the molecule.